Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5–100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1–1,000 nM for all MTXPGn (R ²?>?0.99). The coefficient of variation ranged from 1–4% for intraday precision and 6–15% for interday precision. Samples were stable for at least 1 month at ?80 °C. Recovery ranged from 98–100%, and the relative matrix-effect varied from 95–99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

Assessment of intracellular methotrexate and methotrexate-polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry

A new ultrafast quantitative and high‐throughput mass spectrometric method using matrix‐assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid‐phase extraction of MTX and MTX‐polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within‐run and between‐run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal‐to‐noise (S/N) ratio = 3:1 in drug‐ free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX‐polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low‐dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10⁶ erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX‐polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients. Copyright © 2011 John Wiley & Sons, Ltd.     

Complexation of divalent metal ions with diols in the presence of anion auxiliary ligands: zinc-induced oxidation of ethylene glycol to glycolaldehyde by consecutive hydride ion and proton shifts

Ternary complexes of the type AH???M(2+)???L(-) (AH?=?diol, including diethylene and triethylene glycol, M?=?Ca, Mn, Fe, Co, Ni, Cu and Zn and auxiliary anion ligand L(-) =?CH(3)COO(-), HCOO(-) and Cl(-)) have been generated in the gas phase by MALDI and ESI, and their dissociation characteristics have been obtained. Use of the auxiliary ligands enables the complexation of AH with the divalent metal ion without AH becoming deprotonated, although A(-)???M(2+) is often also generated in the ion source or after MS/MS. For M?=?Ca, dissociation occurs to AH?+?M(2+)???L(-) and/or to A(-)???M(2+) + LH, the latter being produced from the H-shifted isomer A(-) ???M(2+)???LH. For a given ligand L(-), the intensity ratio of these processes can be interpreted (barring reverse energy barriers) in terms of the quantity PA(A(-)) - Ca(aff) (A(-)), where PA is the proton affinity and Ca(aff) is the calcium ion affinity. Deuterium labeling shows that the complex ion HOCH(2)CH(2) OH???Zn(2+)???(-)OOCCH(3), in addition to losing acetic acid (60 Da), also eliminates glycolaldehyde (HOCH(2)CH=O, also 60 Da); it is proposed that these reactions commence with a hydride ion shift to produce the ion-dipole complex HOCH(2)CHOH(+)??? HZnOOCCH(3), which then undergoes proton transfer and dissociation to HOCH(2)CH=O?+?HZn(+)???O?=?C(OH)CH(3). In this reaction, ethylene glycol is oxidized by consecutive hydride ion and proton shifts. A minor process leads to loss of the isomeric species HOCH=CHOH.     

Complexation of divalent metal ions with diols in the presence of anion auxiliary ligands: zinc‐induced oxidation of ethylene glycol to glycolaldehyde by consecutive hydride ion and proton shifts

Ternary complexes of the type AH???M²⁺???L^– (AH = diol, including diethylene and triethylene glycol, M = Ca, Mn, Fe, Co, Ni, Cu and Zn and auxiliary anion ligand L^– = CH₃COO^–, HCOO^– and Cl^–) have been generated in the gas phase by MALDI and ESI, and their dissociation characteristics have been obtained. Use of the auxiliary ligands enables the complexation of AH with the divalent metal ion without AH becoming deprotonated, although A^–???M²⁺ is often also generated in the ion source or after MS/MS. For M = Ca, dissociation occurs to AH + M²⁺???L^– and/or to A^–???M²⁺ + LH, the latter being produced from the H‐shifted isomer A^–???M²⁺???LH. For a given ligand L^–, the intensity ratio of these processes can be interpreted (barring reverse energy barriers) in terms of the quantity PA(A^–) – Ca_aff(A^–), where PA is the proton affinity and Ca_aff is the calcium ion affinity. Deuterium labeling shows that the complex ion HOCH₂CH₂OH???Zn²⁺???^–OOCCH₃, in addition to losing acetic acid (60 Da), also eliminates glycolaldehyde (HOCH₂CH=O, also 60 Da); it is proposed that these reactions commence with a hydride ion shift to produce the ion–dipole complex HOCH₂CHOH⁺??? HZnOOCCH₃, which then undergoes proton transfer and dissociation to HOCH₂CH=O + HZn⁺???O = C(OH)CH₃. In this reaction, ethylene glycol is oxidized by consecutive hydride ion and proton shifts. A minor process leads to loss of the isomeric species HOCH=CHOH. Copyright © 2012 John Wiley & Sons, Ltd.     

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.     

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3)?pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.     

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®.