Measurement of the DNA alkylating agents busulfan and melphalan in human plasma by mass spectrometry.

Busulfan and melphalan are cytotoxic DNA alkylating agents that are used in many hematopoietic stem cell transplantation (HCT) conditioning regimens. We report the development of an assay using turbulent flow liquid chromatography (TFLC) and tandem mass spectrometry to simultaneously measure the concentration of busulfan (Bu) and melphalan (Mel) in human plasma. The method involves precipitating proteins in the plasma specimen with an organic solvent containing deuterated internal standards of both compounds. Following centrifugation, an aliquot of the supernatant was injected into the TFLC mass spectrometry system operated in the positive ion mode. The analytical measurement range for both compounds was 10-5000 ng/mL, and with validated dilutions the reportable range was extended to 25,000 ng/mL. Intra-day and inter-day (n = 20 day) precision studies showed a coefficient of variation (CV) of <7% at several concentrations across the measurement range. To determine accuracy recovery studies were performed at several concentrations spanning the measurement range. Recoveries for both compounds were between 98 and 103%. Additionally, busulfan was compared with an existing assay and showed excellent correlation. Experiments were conducted to rule out matrix effects, carryover and interference from endogenous substances. The validated clinically reportable range (CRR) and assay precision will allow this assay to be used clinically to monitor and adjust Mel and Bu levels to ensure better therapeutic outcomes and also to support clinical trials aimed at better defining therapeutic ranges.

A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry.

A simple and sensitive method for the detection of methylmalonic acid in serum without derivatization has been developed. This method implements protein precipitation using methanol followed by additional sample clean up by turbulent flow liquid chromatography (TFLC). The sample was directly injected into the turbulent flow liquid chromatography tandem mass spectrometry system (TFLC-MS/MS) for online extraction followed by HPLC separation. The eluent was transferred to the mass spectrometer and ionized by heated electrospray negative ionization (HESI) and the analyte was quantified using a six-point calibration curve. The validated analytical measurement range (AMR) is 30-1,000 nMol/L. Dilutions of 10 and 200-fold were validated giving a clinical reportable range (CRR) of 30-200,000 nMol/L. The between-day and within-day imprecision values at concentrations spanning the AMR were less than 15%. This method was compared to an established LC-MS/MS method at a CLIA certified national reference laboratory and shows an excellent correlation with our TFLC-MS/MS method.

Quantification of Docetaxel in Serum Using Turbulent Flow Liquid Chromatography Electrospray Tandem Mass Spectrometry (TFC-HPLC-ESI-MS/MS).

Docetaxel is a second-generation taxane and is used clinically as an anti-neoplastic agent in cancer chemotherapy via an anti-mitotic mechanism. Its efficacy is limited to a narrow therapeutic window. Inappropriately high concentrations may cause erythema, fluid retention, nausea, diarrhea, and neutropenia. As a result, dosing recommendations have changed from high dosage loading every 3 weeks to lower dosage loading weekly. We describe a method that can be used for therapeutic drug monitoring of docetaxel levels using turbulent flow liquid chromatography electrospray tandem mass spectrometry (TFC-HPLC-ESI-MS/MS). The method is rapid, requiring only 6.3 min per analytical run following a simple protein crash. The method requires only 100 µL of serum. Concentrations of docetaxel were quantified by a calibration curve relating the peak-area ratio of docetaxel to a deuterated internal standard (docetaxel-D9). The method was linear from 7.8 to 1000 ng/mL, with imprecision ≤6.2 %.

Quantification of Hydroxychloroquine in Blood Using Turbulent Flow Liquid Chromatography-Tandem Mass Spectrometry (TFLC-MS/MS).

Hydroxychloroquine (HQ) is used routinely in the treatment of autoimmune disorders such as rheumatoid arthritis and lupus erythematosus. Issues such as marked pharmacokinetic variability and patient non-compliance make therapeutic drug monitoring of HQ a useful tool for management of patients taking this drug. Quantitative measurements of HQ may aid in identifying poor efficacy as well as provide reliable information to distinguish patient non-compliance from refractory disease. We describe a rapid 7-min assay for the accurate and precise measurement of HQ concentrations in 100 µL samples of human blood using turbulent flow liquid chromatography coupled to tandem mass spectrometry. HQ is isolated from EDTA whole blood after a simple extraction with its deuterated analog, hydroxychloroquine-d4, in 0.33 M perchloric acid. Samples are then centrifuged and injected onto the TFLC-MS/MS system. Quantification is performed using a nine-point calibration curve that is linear over a wide range (15.7-4000 ng/mL) with precisions of <5 %.

Development of an Assay for Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS.

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 µL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to 1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.

Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum.

A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100µL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.