New steps in infliximab therapeutic drug monitoring in patients with inflammatory bowel diseases.

AIMS: Therapeutic drug monitoring (TDM) of infliximab (IFX) appears to be beneficial for patients with inflammatory bowel disease (IBD). However, the recommended target concentrations depend partly on the method used to quantify IFX. Since we recently developed a liquid chromatography-tandem mass spectrometry method to quantify IFX, we aimed to determine IFX trough concentrations (Cmin) associated with biological remission. METHODS: We retrospectively measured IFX Cmin in sera from 55 patients with IBD, on IFX maintenance therapy, and for whom demographic, biological and clinical data were collected from medical records. A threshold of IFX Cmin associated with biological remission (defined by C-reactive protein < 5 mg l-1 and faecal calprotectin <150 µg g-1 ) was determined using receiver operating characteristics analysis. RESULTS: IFX Cmin ranged from <1 mg l-1 to 57.2 mg l-1 . IFX Cmin were higher (P = 0.038) in patients with biological remission and a cut-off of IFX Cmin set to 6.2 mg l-1 was associated with biological remission (sensitivity = 0.75, 95% confidence interval 0.58-0.75; specificity = 0.61, 95% confidence interval 0.39-0.83). CONCLUSION: Liquid chromatography-tandem mass spectrometry measurement of IFX Cmin and the determination of a new threshold of IFX Cmin associated with biological remission are new steps towards IFX treatment personalization in patients with IBD.

Simultaneous Quantification of Adalimumab and Infliximab in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Adalimumab (ADA) and infliximab (IFX) are therapeutic monoclonal antibodies targeting tumor necrosis factor-alpha (TNFα). They are used to treat inflammatory diseases. Clinical trials have suggested that therapeutic drug monitoring for ADA or IFX could improve treatment response and cost effectiveness. However, ADA and IFX were quantified by ELISA in all these studies, and the discrepancies between the results obtained raise questions about their reliability. We describe here the validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADA and IFX in human samples. METHODS: Full-length antibodies labeled with stable isotopes were added to plasma samples as an internal standard. Samples were then prepared using Mass Spectrometry Immunoassay followed by trypsin digestion before ADA and IFX quantification by LC-MS/MS. ADA and IFX were quantified in serum from patients treated with ADA (n = 21) or IFX (n = 22), and the concentrations obtained were compared with those obtained with a commercial ELISA kit. RESULTS: The chromatography run lasted 8.6 minutes, and the quantification range was 1-26 mg/L. The method was reproducible, repeatable, and accurate. For both levels of internal quality control, for ADA and IFX, interday and intraday coefficients of variation and accuracies were all within 15%, in accordance with FDA recommendations. No significant cross-contamination effect was noted. Good agreement was found between LC-MS/MS and ELISA results, for both ADA and IFX. CONCLUSIONS: This LC-MS/MS method can be used for the quantification of ADA and IFX in a single analytical run and for the optimization of LC-MS/MS resource use in clinical pharmacology laboratories.

Infliximab quantitation in human plasma by liquid chromatography-tandem mass spectrometry: towards a standardization of the methods?

Infliximab (IFX) is a chimeric monoclonal antibody targeting tumor necrosis factor-alpha. It is currently approved for the treatment of certain rheumatic diseases or inflammatory bowel diseases. Clinical studies have suggested that monitoring IFX concentrations could improve treatment response. However, in most studies, IFX was quantified using ELISA assays, the resulting discrepancies of which raised concerns about their reliability. Here, we describe the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for IFX quantification in human plasma. Full-length stable-isotope-labeled antibody (SIL-IFX) was added to plasma samples as internal standard. Samples were then prepared using Mass Spectrometry Immuno Assay (MSIA™) followed by trypsin digestion and submitted to multiple reaction monitoring (MRM) for quantification of IFX. The chromatographic run lasted 13 min. The range of quantification was 1 to 26 mg/L. For two internal quality controls spiked with 6 and 12 mg/L of IFX, the method was reproducible (coefficients of variation (CV%): 12.7 and 2.1), repeatable (intra-day CV%: 5.5 and 5.0), and accurate (inter-day and intra-day deviations from nominal values: +6.4 to +3.7 % and 5.5 to 9.2 %, respectively). There was no cross - contamination effect. Samples from 45 patients treated with IFX were retrospectively analyzed by LC-MS/MS and results were compared to those obtained with an in-house ELISA assay and the commercial Lisa Tracker® method. Good agreement was found between LC-MS/MS and in-house ELISA (mean underestimation of 13 % for in-house ELISA), but a significant bias was found with commercial ELISA (mean underestimation of 136 % for commercial ELISA). This method will make it possible to standardize IFX quantification between laboratories. Graphical Abstract Interassay comparison of the three methods: LC-MS/MS vs inhouse ELISA assay or vs Lisa Tracker® ELISA assays, Passing & Bablok (a) and Bland & Altman (b) for the comparison of LC-MS/MS vs in-house ELISA assay; Passing & Bablok

Trends in drivers testing positive for drugs of abuse in oral fluid from 2018 to 2021 in France.

BACKGROUND: Driving under the influence of drugs (DUID) is a risk factor for traffic accidents. The testing of oral fluid by roadside immunochromatography and laboratory-confirmed chromatography coupled to mass spectrometry (LC-MS/MS) analysis to detect drug abuse has increased in France. The aim of this study was to describe the trends observed in drivers testing positive for illicit drugs in oral fluid and to investigate the concordance between the two analytical methods used. METHODS: We received for confirmation 3051 oral fluid samples from drivers who had tested positive at the roadside with a Drugwipe-5S® device between 2018 and 2021 around Grenoble, France. Samples were collected with FLOQSwab® and analyzed by LC-MS/MS (THC, amphetamine, methamphetamine, MDMA and MDA, MDEA, cocaine and benzoylecgonine, morphine and 6-monoacetylmorphine) at Grenoble Alpes University Hospital, France. Binomial logistic regression was performed to evaluate consumption trends. RESULTS: Most of the drivers were men (93.2%), with a median age of 26 years (range: 14-66 years). Cannabis (94.6%) cocaine (17.5%) and MDMA (2.5%) were the drugs most frequently detected. Poly-drug use was observed in 17.3% of drivers and involved cannabis and cocaine in 85.3% of these drivers. Poly-drug use was more frequent among drivers over the age of 32 years (OR, 3.48; 95% CI, 2.59-4.68; p ≤ .001), as was cocaine use (OR, 5.15; 95% CI, 3.75-7.08; p ≤ .001). The frequency of positive tests for amphetamines was higher in women than in men (OR, 2.53; 95% CI, 1.50-4.27; p ≤ .001). The positive predictive value of Drugwipe-5S was 98.2% for cannabis, 22.6% for amphetamines, 75.4% for cocaine and 17.3% for opiates. At least one discrepancy between Drugwipe-5S® and LC-MS/MS results was observed for 22.3% of the samples tested. CONCLUSION: We report recent trends for drivers testing positive for illicit drugs in oral fluid in France. Cannabis was the most prevalent drug of abuse identified, suggesting that a general prevention program might be useful. Our results also highlight the need for LC-MS/MS confirmation when screening oral fluid for drugs of abuse.

Quantification of belatacept by liquid chromatography-tandem mass spectrometry in human plasma: Application to a pharmacokinetic study in renal transplant recipients.

Therapeutic drug monitoring is the cornerstone of immunosuppressive treatment in transplantation. The immunosuppressive drugs used in kidney transplant patients are mostly comprised of biologics, including therapeutic monoclonal antibodies (mAbs) and fusion proteins. Therefore, a specific and sensitive analytical technique that can universally quantify mAbs, as well as fusion proteins, is essential for clinical pharmacokinetics studies. In this short communication, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for quantification of the fusion protein belatacept in the plasma of kidney-transplant patients. Sample preparation was based on our previously published and implementable electrospray ionization LC-MS/MS method that allows the simultaneous quantification of seven mAbs. Immunocapture was made possible by the Fc domain of belatacept and identification/quantification by the choice of MRM transitions of peptides. The temporal evolution of the belatacept concentration after intravenous infusion and inter-individual variability of trough concentrations were assessed in 17 human plasma samples. The belatacept calibration curves were linear from 1 to 200 mg.L-1 and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Residual belatacept concentrations (n = 8) ranged from 5.1 to 15.0 mg.L-1, with a median of 8.9 mg.L-1 and an inter-individual CV of 33.0%. Our generic LC-MS/MS method allows the quantification of fusion proteins, such as belatacept, and could be used for therapeutic drug monitoring. This method provides a useful tool to study the intra-patient variability of belatacept and the association between belatacept exposure and its therapeutic effects.

Leukotriene B4 pathway activation and atherosclerosis in obstructive sleep apnea.

Leukotriene B(4) (LTB(4)) production increases in obstructive sleep apnea syndrome (OSA) and is linked to early vascular remodeling, the mechanism of which is unknown. The objective of this study was to to determine the molecular mechanisms of LTB(4) pathway activation in polymorphonuclear cells (PMNs) and early vascular remodeling in OSA and the specific contribution of intermittent hypoxia (IH). PMNs were isolated from 120 OSA patients and 33 healthy subjects and used for measurements of LTB(4) production, determination of mRNA and protein expression levels, or exposed for four cycles of in vitro IH. PMNs derived from OSA patients exhibited increased LTB(4) production, for which apnea-hypopnea index was an independent predictor (P=0.042). 5-Lipoxygenase-activating protein (FLAP) mRNA and protein increased significantly in PMNs from OSA patients versus controls and were associated with carotid luminal diameter and intima-media thickness. LTB(4) (10 ng/ml) increased IL-6 (P=0.006) and MCP-1 (P=0.002) production in OSA patient monocytes. In vitro exposure of PMNs from controls to IH enhanced FLAP mRNA levels (P= 0.027) and induced a 2.7-fold increase (P=0.028) in LTB(4) secretion compared with PMNs exposed to normoxia. In conclusion, upregulation of FLAP in PMNs in response to IH may participate in early vascular remodeling in OSA patients, suggesting FLAP as a potential therapeutic target for the cardiovascular morbidity associated with OSA.

A simple and easy-to-perform liquid chromatography-mass spectrometry method for the quantification of tacrolimus and its metabolites in human whole blood. Application to the determination of metabolic ratios in kidney transplant patients.

Tacrolimus is the cornerstone of immunosuppressive therapy in solid organ transplantation and its blood concentrations are routinely monitored. Tacrolimus is extensively metabolized into metabolites that are supposed to be nephrotoxic. Yet, few analytical methods have been described to simultaneously quantify tacrolimus and its main metabolites. We developed and validated a simple liquid chromatography-mass spectrometry method for the quantification of tacrolimus and its three desmethylated metabolites, 13-O, 15-O, and 31-O-desmethylated tacrolimus (M-I, M-III, and M-II respectively) in human whole blood. Protein precipitation of 50 µL of whole blood with 100 µL methanol and zinc sulfate was used as a single-extraction procedure. Tacrolimus and its metabolites were quantified using electrospray ionization-triple quadrupole mass spectrometry in combination with selected reaction monitoring detection in the positive ionization mode. The method was validated following FDA recommendations. This method was precise (intra- and inter-assay coefficients of variation: 2.88-7.81% and 3.96-12.10% for low and high levels of internal quality controls, respectively) and accurate (intra- and inter-assay biases: -1.67-10.30%, and -0.77--9.36%, respectively). In adult kidney transplant patients who were treated with tacrolimus prolonged release formulation, the median (10th-90th percentiles) trough concentrations (n = 16) of tacrolimus, M-I, and M-III were 5.85 (3.37-7.09), 0.100 (0.037-0.168), 0.051 (0.03-0.104), respectively. M-II was measured in only 2 trough samples. The metabolic ratios M-I/tacrolimus and M-III/tacrolimus were 0.017 (0.009-0.027) and 0.009 (0.006-0.015) when measured on trough concentration and 0.022 (0.011-0.037) and 0.008 (0.006-0.015) when measured on area under the curves 0-24 h. This method is a suitable and easy-to-perform tool for future pharmacokinetic-pharmacodynamics studies investigating the importance of tacrolimus and its metabolites blood exposure for solid organ graft survival.

Variability of rituximab and tocilizumab trough concentrations in patients with rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) are eligible for treatment with therapeutic monoclonal antibodies (mAbs) that target tumor necrosis factor α (TNFα), as well as others, such as rituximab (RTX) and tocilizumab (TCZ). Although pharmacokinetic variability and the link between concentration-clinical response of anti-TNFα mAbs have been well-described, little is known about RTX and TCZ. We aimed to evaluate the variability of RTX and TCZ serum concentrations in RA patients treated in second-line and the relationship between RTX/TCZ concentrations and the clinical response. Serum mAb trough concentrations of RA patients treated with RTX (n = 35) or TCZ (n = 46) were determined at week 24 by liquid chromatography-tandem mass spectrometry. The clinical response was assessed at week 24 by the change in the disease activity score in the 28 joints-erythrocyte sedimentation rate from baseline (ΔDAS28-Erythrocyte Sedimentation Rate) and according to the European League Against Rheumatism (EULAR) recommendations. RTX and TCZ trough concentrations were highly variable, with a coefficient of variation of 171.3% for RTX (median [10th-90th percentiles]: <1.0 µg/mL [<1.0-5.1]) and 132.6% for TCZ (median [10th-90th percentiles]: 5.4 µg/mL [<1.0-27.8]). Univariate analysis did not identify any determinants of such variability, except cotreatment with methotrexate, which was associated with lower RTX concentrations (P = 0.03). The response to treatment was not related to the RTX or TCZ trough concentration. RTX and TCZ trough concentrations at 24 weeks were highly variable in RA patients treated in the second line, without any link concentration-clinical response having been demonstrated.

Simultaneous quantification of rituximab and eculizumab in human plasma by liquid chromatography-tandem mass spectrometry and comparison with rituximab ELISA kits.

OBJECTIVES: Specific and sensitive analytical techniques to quantify therapeutic monoclonal antibodies (mAbs) are required for therapeutic drug monitoring. The quantification of mAbs has been historically performed using enzyme-linked immunosorbent assays (ELISAs), for which the limitations in terms of specificity have led to the development of alternative analytical strategies. METHODS: Here, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous quantification of rituximab (RTX - anti-CD20) and eculizumab (ECU - anti-C5). Sample preparation was based on our previously published method, using protein G purification and trypsin digestion. A new specific peptide for RTX, containing an N-terminal pyroglutamine and a trypsin miss-cleavage, enables better sensitivity, while peptide of ECU was chosen thanks to an in silico trypsin digestion and the Skyline® software. Full-length stable-isotope-labeled adalimumab was added to plasma samples as an internal standard. RTX in 50 human serum samples was quantified by LC-MS/MS and the concentrations obtained compared to those obtained with two commercial ELISA kits (Lisa Tracker® and Promonitor®). RESULTS: Calibration curves were linear from 1 to 200 µg.mL-1 for RTX and 5 to 200 µg.mL-1 for ECU, and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Comparison of the LC-MS/MS method with ELISA showed a negligible bias with the Lisa Tracker® kit (4%), but significant bias with the Promonitor® assay (mean underestimation of 69% for the Promonitor® assay). CONCLUSIONS: This new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring.

Feasibility of ribavirin therapeutic drug monitoring in hepatitis C.

Ribavirin, a nucleoside analog, is administered in combination with interferon to patients with chronic hepatitis C. To evaluate the feasibility of ribavirin therapeutic drug monitoring, we investigated the influence of blood collection and preanalytical conditions on ribavirin concentrations and compared the results obtained from interlaboratory blind tests by 3 laboratories using different analytical techniques. On 3 occasions, blank serum samples spiked with ribavirin and pooled serum samples from patients on ribavirin were sent to the 3 laboratories. Two analytical techniques were based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and 1 on high-performance liquid chromatography with UV detection (HPLC-UV), with protein precipitation or solid-phase extraction, all validated according to international guidelines. Inter- and intra-batch mean relative errors ranged from -7.4% to +10.3% and from -10.3% to +7.4%, respectively. Relative standard deviations were <13.5% and <10.6%, respectively. Linearity, assessed blindly, between 125 and 4550 ng/mL was excellent (r > 0.991) for all 3 methods. The 2 LC-MS/MS techniques were slightly less precise and accurate than HPLC-UV, perhaps because the internal standard used was not a ribavirin isotope. Accurate and precise LC-MS/MS and HPLC-UV methods developed in 3 different laboratories provided excellent and consistent results to blind tests for ribavirin determination in spiked serum samples and pools of serum samples from patients with chronic hepatitis C virus infection.

A multiplex liquid chromatography tandem mass spectrometry method for the quantification of seven therapeutic monoclonal antibodies: Application for adalimumab therapeutic drug monitoring in patients with Crohn's disease.

The use of therapeutic monoclonal antibodies (mAbs) is steadily increasing. Previous studies have reported the clinical interest of mAb therapeutic-drug monitoring (TDM), including that of adalimumab, for patients with Crohn's disease (CD). Proof of concept mAb-quantification studies by liquid chromatography mass spectrometry (LC-MS/MS) have been published, but a specific and reliable routine-suited multiplex quantification method is still needed to facilitate mAb TDM. We describe an electrospray ionization LC-MS/MS method for the simultaneous quantification of seven mAbs (adalimumab, cetuximab, infliximab, rituximab, secukinumab, tocilizumab, and trastuzumab) in human plasma. Sample preparation was performed using protein-G purification and trypsin digestion to obtain proteotypic peptides. We retrospectively measured the adalimumab concentration in 65 plasma samples from 56 CD patients and determined the adalimumab therapeutic cut-off concentration associated with biological remission. Calibration curves were linear from 1 to 100 µg mL-1, except for rituximab (5-100 µg mL-1). This method was reproducible, repeatable, and accurate (coefficient of variation and bias < 20%), with no cross contamination. Adalimumab concentrations were significantly higher (p = 0.0198) for patients with biological remission (median: 11.3 µg mL-1 [4.6; 18.3]) than that for patients without a biological response (9.5 µg mL-1 [3.94;17.0]). An adalimumab cut-off concentration of 8.0 µg mL-1 correctly discriminated patients with or without biological remission (sensitivity: 74.1%, specificity: 57.9%). This validated LC-MS/MS routine-suited method is the first allowing simultaneous quantification of up to seven mAbs acting against different pharmacological targets. It opens the field of TDM to numerous mAbs.

Lethal cerebral hemorrhage after ticagrelor intoxication: a specific antidote is urgently needed.

BACKGROUND: Ticagrelor is a direct and reversible competitive antagonist of the P2Y12 receptor and inhibits platelet activation. Although adverse bleeding is common, fatal intoxication has never been documented. CASE DESCRIPTION: A 47-year-old man died from a severe cerebral hemorrhage secondary to a fall and cranial trauma 4 d after the massive intake of ticagrelor. Iterative platelet transfusions did not improve his condition. Toxicological analyses by liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed high plasma concentrations of ticagrelor (3343 µg/L) and its active metabolite AR-C124910XX (656 µg/L) 10 h after intake. The approximate ingested dose was extrapolated to 1677 mg. Assessment of ADP-induced platelet aggregation and platelet Vasodilator Stimulated Phosphoprotein phosphorylation (VASP), 2 and 3 d after admission, respectively, showed the persistence of platelet inhibition. DISCUSSION: To the best of our knowledge, no prior fatal cases have been reported and documented with both ticagrelor and AR-C124910XX concentrations. Our findings highlight the need for a specific antidote to manage such complications resulting from ticagrelor overdose.

Simultaneous quantitation of azole antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional high-performance liquid chromatography-tandem mass spectrometry.

High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard analytical technique for therapeutic drug monitoring (TDM). A rapid LC-MS/MS method was developed for simultaneous quantitation of 3 antifungals and one active metabolite (posaconazole, voriconazole, itraconazole, and hydroxy-itraconazole), 5 antibiotics (daptomycin, ciprofloxacin, oxacillin, levofloxacin, and rifampicin), an antineoplastic agent (imatinib), and an antiretroviral (raltegravir) in human plasma. Protein precipitation of 10 µL of plasma with acetonitrile was used as a single-extraction procedure. After 2-dimensional LC, all drugs were quantified by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated per FDA recommendations including the study of extraction recovery (from 79.3% to 105.9%) and matrix effect via ion suppression/enhancement phenomenon. This method is precise (intra- and inter-assay coefficients of variation of 1.95-12.77%, 2.56-8.16% and 2.12-11.38% for low, medium and high levels of internal quality controls respectively) and accurate (intra- and inter-assay biases of 0.19-12.67%, 0.04 to -12.17% and 0.22-12.98% respectively). This method is an efficient tool for routine TDM and optimization of laboratory resource utilization.

Ultra-fast cyclosporin A quantitation in whole blood by Laser Diode Thermal Desorption-tandem mass spectrometry; comparison with High Performance Liquid Chromatography-tandem mass spectrometry.

In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization-tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM). A simple protein precipitation step was used prior to depositing 5 µL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS. The laser pattern was set to ramp from 0 to 45% laser power within 2 s. The APCI parameters were set to negative needle voltage (-2 µA), carrier gas temperature (30°C) and air flow rate (3 L min(-1)). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of -40 V. The analysis was achieved within 9 s from sample to sample. The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5 ng mL(-1) (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965 ng mL(-1) was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC-ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982-0.991). The Passing-Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.

Lack of specificity for the analysis of raltegravir using online sample clean-up liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: Raltegravir is the first antiretroviral agent to target the human immunodeficiency virus-1 (HIV-1) integrase. It is indicated, in association with other antiretrovirals, in the treatment of acquired immunodeficiency syndrome (AIDS) in antiretroviral treatment-experienced adult patients with viral resistance. To evaluate the feasibility of raltegravir therapeutic drug monitoring, we developed a rapid and specific analytical method for the quantification of raltegravir in human plasma by online sample clean-up liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: After protein precipitation (with 100 microL of acetonitrile/methanol (50/50)) of 25 microL of plasma, fast online matrix-clean-up was performed using a column switching program. The chromatographic step was optimized to separate raltegravir and its glucuronide metabolite (G-raltegravir). Multiple reaction monitoring (MRM) was used for detection of raltegravir and G-raltegravir. In the absence of G-raltegravir standard, G-raltegravir identification was confirmed by beta-glucuronidase pre-treatment. RESULTS: A total analysis of 3.8 min was needed to separate raltegravir to G-raltegravir. The method was linear between 10 and 3000 ng/mL for raltegravir. Analytical recovery was 94+/-1%. Variation coefficients ranged between 5% and 8.4%. Pre-treatment of plasma from a patient under raltegravir treatment with beta-glucuronidase suppressed G-raltegravir peak. CONCLUSION: We describe a fast online LC-MS/MS assay that is valid and reliable for the quantification of raltegravir, despite the lack of specificity that could occur in MRM scanning mode experiments.