Quantitation of Estradiol and Testosterone in Serum Using LC-MS/MS.

Adult and pediatric endocrinology and oncology often requires measuring serum estrogens and testosterone at very low concentrations. Conventional immunoassay methods often lack the required performance to meet this analytical need, and mass spectrometry techniques must be employed. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum, utilizing commercially available calibrators and without the need for chemical derivatization. Serum samples, after the addition of an internal standard, are combined with a hexane:ethyl acetate extraction solution. The samples are vortexed, and the organic layer is decanted into a clean sample tube and evaporated to dryness under a stream of nitrogen. The samples are reconstituted in a water:methanol solution and separated chromatographically using a reversed-phase HPLC column. Subsequent mass spectrometry is performed using both positive ion mode for Te and negative ion mode for E2.

A prospective study of dysgeusia and related symptoms in patients with multiple myeloma after autologous hematopoietic cell transplantation.

BACKGROUND: Dysgeusia is a common but understudied complication in patients undergoing autologous hematopoietic cell transplantation (auto-HCT). We assessed the feasibility of using chemical gustometry (CG) to measure dysgeusia and explored its associations with symptom burden, nutrition, chemotherapy pharmacokinetics (PK), and the oral microbiome. METHODS: We conducted a single-center, prospective feasibility study (NCT03276481) of patients with multiple myeloma undergoing auto-HCT. CG was performed longitudinally testing five flavors (sweet, sour, salty, bitter, umami) to calculate a total taste score (maximum score, 30). We measured caloric intake and patient-reported symptoms, assessing their correlation with oral microbiota composition and salivary and blood melphalan PK exposure. RESULTS: Among all 45 patients, 39 (87%) completed at least four (>60%) and 22 (49%) completed all six CG assessments. Median total CG scores remained stable over time but were lowest at day +7 (27, range 24-30) with recovery by day +100. Symptom burden was highest by day +10 (area under the curve, 2.9; range, 1.0-4.6) corresponding with the lowest median overall caloric intake (1624 kcal; range, 1345-2267). Higher serum/salivary melphalan levels correlated with higher patient-reported dysgeusia and lower caloric intake. Oral microbiota α-diversity was stable early and increased slightly by day +100. CONCLUSIONS: Assessment of dysgeusia by CG is feasible after auto-HCT. Most dysgeusia, symptom burden, and lowest caloric intake occurred during the blood count nadir. Higher melphalan concentrations correlated with more dysgeusia and poorer caloric intake. Future studies will aim to modulate melphalan exposure by PK-targeted dosing and characterize patient taste preferences to personalize diets for improved nutritional intake. LAY SUMMARY: Taste changes after cancer treatments are very common. We used chemical gustometry (taste testing) to study taste changes and to better understand why patients with multiple myeloma experience this symptom after autologous hematopoietic cell transplantation. We found that taste testing was feasible, taste changes peaked when blood counts were lowest, and most patients recovered their taste by 100 days after transplantation. Taste changes correlated with lower food intake and with higher levels of chemotherapy in the body. Future work will focus on using personalized chemotherapy doses to reduce taste changes and to match patients' individual taste preferences with their diets.

Evaluation of Melphalan Exposure in Lymphoma Patients Undergoing BEAM and Autologous Hematopoietic Cell Transplantation.

High-dose melphalan is one of the main cytotoxic DNA alkylating agents and is used in many transplantation conditioning regimens. Studies have shown a wide range of drug exposure when a traditional weight-based dose of melphalan is used. The optimal melphalan dose in BEAM (carmustine, etoposide, cytarabine, and melphalan), which results in maximum efficacy with acceptable toxicity, is unknown. In this pharmacokinetic (PK) analysis of 105 patients with lymphoma undergoing treatment with BEAM and autologous hematopoietic cell transplantation, we initially estimated melphalan exposure as area under the curve (AUC) by a noncompartmental analysis and subsequently compared it with a newly developed 2-compartment population-PK model. The 2 models correlated closely with each other. We found that the traditional fixed weight-based dosing of propylene glycol-free (captisol-enabled) melphalan in BEAM results in a wide variation in exposure as estimated by both models. Higher melphalan exposure was significantly associated with increased metabolic toxicities but did not seem to impact progression-free survival. Although our study suggests a melphalan AUC of 8 mg·h/L as a potential target in BEAM, larger prospective studies using personalized PK-directed melphalan dosing are needed to determine the optimal melphalan exposure in lymphomas.

Routine use of low-dose glucarpidase following high-dose methotrexate in adult patients with CNS lymphoma: an open-label, multi-center phase I study.

BACKGROUND: High-dose methotrexate (HD-MTX) has broad use in the treatment of central nervous system (CNS) malignancies but confers significant toxicity without inpatient hydration and monitoring. Glucarpidase is a bacterial recombinant enzyme dosed at 50 units (u)/kg, resulting in rapid systemic MTX clearance. The aim of this study was to demonstrate feasibility of low-dose glucarpidase to facilitate MTX clearance in patients with CNS lymphoma (CNSL). METHODS: Eight CNSL patients received HD-MTX 3 or 6 g/m2 and glucarpidase 2000 or 1000u 24 h later. Treatments repeated every 2 weeks up to 8 cycles. RESULTS: Fifty-five treatments were administered. Glucarpidase 2000u yielded > 95% reduction in plasma MTX within 15 min following 33/34 doses (97.1%) and glucarpidase 1000u yielded > 95% reduction following 15/20 doses (75%). Anti-glucarpidase antibodies developed in 4 patients and were associated with MTX rebound. In CSF, glucarpidase was not detected and MTX levels remained cytotoxic after 1 (3299.5 nmol/L, n = 8) and 6 h (1254.7 nmol/L, n = 7). Treatment was safe and well-tolerated. Radiographic responses in 6 of 8 patients (75%) were as expected following MTX-based therapy. CONCLUSIONS: This study demonstrates feasibility of planned-use low-dose glucarpidase for MTX clearance and supports the hypothesis that glucarpidase does not impact MTX efficacy in the CNS. CLINICAL TRIAL REGISTRATION: NCT03684980 (Registration date 26/09/2018).

Population Pharmacokinetics of Melphalan in a Large Cohort of Autologous and Allogeneic Hematopoietic Cell Transplantation Recipients: Towards Individualized Dosing Regimens.

BACKGROUND AND OBJECTIVES: High-dose melphalan is an integral part of conditioning chemotherapy prior to both autologous and allogeneic hematopoietic cell transplantation. While underexposure may lead to relapse, overexposure may lead to toxicities include mucositis, diarrhea, bone marrow suppression, and rarely sinusoidal obstruction syndrome. In this study, we describe the population pharmacokinetics of high-dose melphalan as a first step towards individualized dosing. METHODS: Melphalan samples were collected in patients receiving an allogeneic or autologous hematopoietic cell transplantation between August 2016 and August 2020 at the Memorial Sloan Kettering Cancer Center. A population-pharmacokinetic model was developed using NONMEM. RESULTS: Based on a total of 3418 samples from 452 patients receiving a median cumulative dose of 140 mg/m2, a two-compartment population-pharmacokinetic model was developed. Fat-free mass was a covariate for clearance, central volume of distribution, and inter-compartmental clearance, while glomerular filtration rate predicted clearance. Simulation studies showed that based on fixed body surface area-based dosing, renal impairment has a higher impact in increasing melphalan exposure compared with obesity. CONCLUSIONS: The proposed model adequately describes the population pharmacokinetics of melphalan in adult patients receiving a hematopoietic cell transplantation. This model can be used to define the therapeutic window of melphalan, and subsequently to develop individualized dosing regimens aiming for that therapeutic window in all patients.

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.

Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS.

A very sensitive LC-MS/MS assay was developed implementing a liquid-liquid extraction step followed by mass spectrometry which was operated in both positive and negative ion modes. The assay was calibrated with readily available commercial calibrators and compared with international reference standards. This data is also presented in "Sensitive Simultaneous Quantitation of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization and Comparison with the CDC HoSt Program" (Schofield et al., 2017). This article includes the comparison of the LC-MS/MS assay with a commonly available chemiluminescencent immunoassay for the quantitation of both estradiol and testosterone. In addition we show baseline separation of estradiol and testosterone from other structurally related and/or isobaric compounds that could potentially interfere with the assay. In addition, various calibrator materials were tested and compared with internationally-recognized reference materials.

Insulin and glucose homeostasis in childhood cancer survivors treated with abdominal radiation: A pilot study.

BACKGROUND: Childhood cancer survivors exposed to abdominal radiation (abdRT) are at increased risk for both insulin-dependent and non-insulin-dependent diabetes. We sought to clarify the pathophysiology of diabetes after abdRT by performing dynamic studies of insulin and glucose and testing for type 1 diabetes-associated autoantibodies. PROCEDURE: Cross-sectional analysis of 2-year childhood cancer survivors treated with abdRT at age ≤21 years who underwent oral glucose tolerance testing and assessment of diabetes-related autoantibodies from December 2014 to September 2016. Prevalence of insulin/glucose derangements, indices of insulin sensitivity/secretion (homeostatic model assessment of insulin resistance [HOMA-IR], whole-body insulin sensitivity, insulinogenic index), autoantibody positivity, and treatment/demographic factors associated with adverse metabolic outcomes were assessed. RESULTS: Among 40 participants previously exposed to abdRT (57.5% male; median age at cancer diagnosis, 3.3 years [range, 0.5-20.1]; median age at study 14.3 years [range, 8.3-49.8]; none with obesity), 9 (22.5%) had glucose derangements (n = 4 with impaired fasting glucose [≥100 mg/dL]; n = 4 with impaired glucose tolerance [2-hour glucose 140-199 mg/dL]; n = 1 with previously unrecognized diabetes [2-hour glucose ≥200 mg/dL]). Three of the four individuals with impaired fasting glucose also had insulin resistance, as measured by HOMA-IR; an additional four subjects with normal glucose tolerance were insulin resistant. The subject with diabetes had normal HOMA-IR. No participant had absolute insulinopenia or >1 positive diabetes-related autoantibody. CONCLUSIONS: This study suggests that radiation-induced damage to the insulin-producing ß-cells is an unlikely explanation for the early derangements in glucose metabolism observed after abdRT. Research into alternative pathways leading to diabetes after abdRT is needed.

Impact of Toxicity on Survival for Older Adult Patients after CD34+ Selected Allogeneic Hematopoietic Stem Cell Transplantation.

Ex vivo CD34+ selection before allogeneic hematopoietic stem cell transplantation (allo-HCT) reduces graft-versus-host disease without increasing relapse but usually requires myeloablative conditioning. We aimed to identify toxicity patterns in older patients and the association with overall survival (OS) and nonrelapse mortality (NRM). We conducted a retrospective analysis of 200 patients who underwent CD34+ selection allo-HCT using the ClinicMACS® system between 2006 and 2012. All grade 3 to 5 toxicities by CTCAE v4.0 were collected. Eighty patients aged ≥ 60 years with a median age of 64 (range, 60 to 73) were compared with 120 patients aged < 60 years. Median follow-up in survivors was 48.2 months. OS and NRM were similar between ages ≥ 60 and <60, with 1-year OS 70% versus 78% (P = .07) and 1-year NRM 23% versus 13% (P = .38), respectively. In patients aged ≥ 60 the most common toxicities by day 100 were metabolic, with a cumulative incidence of 88% (95% CI, 78% to 93%), infectious 84% (95% CI, 73% to 90%), hematologic 80% (95% CI, 69% to 87%), oral/gastrointestinal (GI) 48% (95% CI, 36% to 58%), cardiovascular (CV) 35% (95% CI, 25% to 46%), and hepatic 25% (95% CI, 16% to 35%). Patients aged ≥ 60 had a higher risk of neurologic (HR, 2.63 [95% CI, 1.45 to 4.78]; P = .001) and CV (HR, 1.65 [95% CI, 1.04 to 2.63]; P = .03) toxicities but a lower risk of oral/GI (HR, .58 [95% CI, .41 to .83]; P = .003) compared with those aged < 60. CV, hepatic, neurologic, pulmonary, and renal toxicities remained independent risk factors for the risk of death and NRM in separate multivariate models adjusting for age and hematopoietic cell transplantation-specific comorbidity index. Overall, the toxicity of a more intense regimen is potentially balanced by the absence of toxicity related to methotrexate and calcineurin inhibitors in older patients. Prospective study of toxicities after allo-HCT in older patients is essential.

Effects of Late Toxicities on Outcomes in Long-Term Survivors of Ex-Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation.

The late adverse events in long-term survivors after myeloablative-conditioned allogeneic hematopoietic cell transplantation (HCT) with ex vivo CD34+ cell selection are not well characterized. Using the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.0, we assessed all grade ≥3 toxicities from the start of conditioning to the date of death, relapse, or last contact in 131 patients who survived >1 year post-HCT, identifying 285 individual toxicities among 17 organ-based toxicity groups. Pretransplantation absolute lymphocyte count >.5 K/µL and serum albumin >4.0 g/dL were associated with a reduced risk of toxicities, death, and nonrelapse mortality (NRM), whereas serum ferritin >1000 ng/mL was associated with an increased risk of toxicities and NRM after 1 year. An HCT Comorbidity Index (HCT-CI) score ≥3 was associated with an increased risk of all-cause death and NRM, but was not associated with a specific increased toxicity risk after 1 year. Patients who incurred more than the median number of toxicities (n = 7) among all patients within the first year subsequently had an increased risk of hematologic, infectious, and metabolic toxicities, as well as an increased risk of NRM and inferior 4-year overall survival (OS) (67% versus 86%; P = .003) after the 1-year landmark. The development of grade II-IV acute graft-versus-host disease (GVHD) within the first year was associated with incurring >7 toxicities within the first year (P = .016), and also with an increased risk of all-cause death and NRM after 1 year. In multivariate models, cardiovascular, hematologic, hepatic, infectious, metabolic, neurologic, and pulmonary toxicities incurred after 1 year were independently associated with increased risk of death and NRM when adjusting for both HCT-CI and grade II-IV acute GVHD within the first year. One-year survivors of ex vivo CD34+ selection had a favorable 4-year OS of 77%, although the development of grade ≥3 toxicities after the first year was associated with poorer outcomes, emphasizing the fundamental importance of improving survivorship efforts that may improve long-term toxicity burden and outcome.

Data from the analytical performance of the Abaxis Piccolo Xpress point of care analyzer in whole blood, serum, and plasma.

The objective of this study was to examine the analytical performance of 14 comprehensive metabolic panel analytes on the Abaxis Piccolo Xpress® Point of Care analyzer in serum, plasma, and whole blood. A method comparison was performed on all three specimen types intended for use on the Piccolo Xpress®: serum, heparinized plasma, and whole blood. This data is also presented in Murata et al. (2015) [1]. This article includes the actual Bland-Altman bias plots of the difference in results obtained for analytes in the comprehensive metabolic panel from the Abaxis Piccolo Xpress and the comparison instrument, the Ortho Vitros.

The Case for Laboratory Developed Procedures: Quality and Positive Impact on Patient Care.

An explosion of knowledge and technology is revolutionizing medicine and patient care. Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures. Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments. A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories.

The Impact of Toxicities on First-Year Outcomes after Ex Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation in Adults with Hematologic Malignancies.

Factors that impact first-year morbidity and mortality in adults undergoing myeloablative allogeneic hematopoietic cell transplantation with ex vivo CD34+ selection have not been previously reported. We assessed all toxicities ≥ grade 3 from the start of conditioning to date of death, relapse, or last contact in 200 patients during the first year after transplantation, identifying 1885 individual toxicities among 17 organ-based toxicity groups. The most prevalent toxicities in the first year were of infectious, metabolic, hematologic, oral/gastrointestinal, hepatic, cardiac, and pulmonary etiologies. Renal complications were minimal. Grades II to IV and III and IV acute GVHD at day 100 were 11.5% and 3%, respectively. In separate multivariate models, cardiovascular, hematologic, hepatic, neurologic, pulmonary, and renal toxicities negatively impacted nonrelapse mortality (NRM) and overall survival during the first year. A higher-than-targeted busulfan level, patient cytomegalovirus seropositivity, and an Hematopoietic Cell Transplantation-Specific Comorbidity Index of ≥3 were associated with increased risk of NRM and all-cause death. Ex vivo CD34+ selection had a favorable 1-year OS of 75% and NRM of 17% and a low incidence of sinusoidal obstruction syndrome. These data establish a benchmark to focus efforts in reducing toxicity burden while improving patient outcomes.

Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC-MS/MS without derivatization and comparison with the CDC HoSt program.

BACKGROUND: Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. METHODS: Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. RESULTS: The lower limits of quantitation (LLOQs) of E2 and Te were 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5-600pg/mL and 1-1,170ng/dL for Te. Assay accuracy was determined both by comparison with a LC-MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N=10) and between-day (N=20) CVs at concentrations spanning the AMR were less than 7% for both analytes. CONCLUSION: We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC-MS/MS without chemical derivatization and using commercially available calibrators.

Topical tacrolimus for the treatment of secondary lymphedema.

Secondary lymphedema, a life-long complication of cancer treatment, currently has no cure. Lymphedema patients have decreased quality of life and recurrent infections with treatments limited to palliative measures. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by promoting tissue fibrosis and inhibiting lymphangiogenesis. Here using mouse models, we show that topical therapy with tacrolimus, an anti-T-cell immunosuppressive drug, is highly effective in preventing lymphedema development and treating established lymphedema. This intervention markedly decreases swelling, T-cell infiltration and tissue fibrosis while significantly increasing formation of lymphatic collaterals with minimal systemic absorption. Animals treated with tacrolimus have markedly improved lymphatic function with increased collecting vessel contraction frequency and decreased dermal backflow. These results have profound implications for lymphedema treatment as topical tacrolimus is FDA-approved for other chronic skin conditions and has an established record of safety and tolerability.

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.

Quantitation of 25-OH-Vitamin-D2 and 25-OH-Vitamin-D3 in Urine Using LC-MS/MS.

Patients with significant proteinuria represent a unique population with respect to vitamin D status due to the urinary losses of vitamin D-binding protein (DBP) to which >99 % of circulating 25-hydroxy vitamin D (25(OH)D) is bound. Low serum concentrations of 25(OH)D have been found in children and adults with nephrotic syndrome (NS). However, previously described assays developed to quantify the magnitude of urinary loss are technically challenging. This chapter describes a simple and sensitive method to quantify 25(OH)D2 and 25(OH)D3 in urine specimens in a single analytical LC-MS/MS analysis. This assay is more sensitive than previously described radioimmunoassays and offers the ability to quantitate both forms of 25-hydroxy vitamin D. The assay involves no chemical derivitization, has a linear measurement range of 20-1500 pg/mL and displays imprecision (CVs) below 7 % at various concentrations across the analytical measurement range.

Simultaneous Quantitation of Lamotrigine, Levetiracetam, 10-Hydroxycarbazepine, Topiramate, and Zonisamide in Serum Using HPLC-MS/MS.

Antiepileptic drugs (AEDs) are a diverse group of pharmacological agents used in the treatment of epileptic seizures. Over the past several decades some new AEDs, including lamotrigine (LTG), levetiracetam (LVA), oxcarbazepine (OXC), topiramate (TOP), and zonisamide (ZNS), have become widely used. This chapter describes a very simple and rapid liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of LVA, ZNS, LTG, TOP, and MHD in human serum. The method requires a very small amount of serum (50 µL) for multiple drug measurements and has a total analysis time of 4 min that makes it well suited for routine clinical analysis of several drugs simultaneously. The imprecision (CVs) measured at various concentrations across the analytical measurement range (AMR) are less than 7% for all analytes. The AMR for each analyte is as follows: LVA (1-100 µg/mL), ZNS (0.8-80 µg/mL), TOP (0.5-50 µg/mL), and 0.6-60 µg/mL for LTG and MHD.

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.