Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7‐hydroxymethotrexate (7‐OH‐MTX) and 2,4‐diamino‐N^10‐methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7‐OH‐MTX, DAMPA and 2,4‐diaminopteroic acid (DAPA) as an internal standard were separated on a reversed‐phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7‐OH‐MTX and DAMPA from plasma at 100 pmol/mL were 81.5 ± 5.4, 82.5 ± 5.3 and 56.2 ± 7.0%, respectively. The limits of quantification for MTX, 7‐OH‐MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within‐ and between‐day variations for MTX, 7‐OH‐MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy. Copyright © 2011 John Wiley & Sons, Ltd.     

A novel high‐performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion‐pairing reversed‐phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates_2–7, in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid‐phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 µL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 µL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long‐chain methotrexate polyglutamates_5–7 and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches. Copyright © 2009 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma: application to a pharmacokinetic study

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid‐phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP_18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow‐gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49–91.0 and 0.40–74.4 ng/mL for MTX and TFB, respectively. The intra‐ and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post‐dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of unbound prednisolone, prednisone and cortisol in human serum and saliva by on-line solid-phase extraction liquid chromatography tandem mass spectrometry and potential implications for drug monitoring of prednisolone and prednisone in saliva

Prednisolone (PLN) and prednisone (PN) are widely used glucocorticoids. Drug monitoring of PLN and PN is not routinely done owing to the need for multiple blood sampling and challenging measurement of unbound PLN and PN in blood. Here we present a robust method for quantification of cortisol, PLN and PN in serum, ultrafiltrate and saliva by on‐line solid‐phase extraction LC‐MS/MS. The method is linear for the three analytes over the range of 6–1400 nmol/L for serum and 2–450 nmol/L for ultrafiltrate and saliva. Within‐run precision of all three analytes was <10% and total precision was <15%. This method was applied to create time–concentration profiles of cortisol, PLN and PN after an oral dose of prednisolone in a healthy volunteer. Salivary levels of PLN correlated well with ultrafiltrate levels (p < 0.01), while this correlation was only marginal for PN (p = 0.052). The PN/PLN ratio was significantly higher in saliva than in ultrafiltrate and serum (p < 0.01). Addition sums of both metabolites in saliva showed excellent correlation with those of ultrafiltrate (p < 0.01). These findings have not been presented before and may have important implications for future studies concerning drug monitoring of PLN and PN in saliva. Copyright © 2011 John Wiley & Sons, Ltd.     

Eco‐friendly ionic liquid assisted capillary electrophoresis and α‐acid glycoprotein‐assisted liquid chromatography for simultaneous determination of anticancer drugs in human fluids

In the current work, two eco‐friendly analytical methods based on capillary electrophoresis (CE) and reversed phase liquid chromatography (RPLC) were developed for simultaneous determination of the most commonly used anticancer drugs for Hodgkin's disease: methotrexate (MTX), vinblastine, chlorambucil and dacarbazine. A background electrolyte (BGE) of 12.5 mmol/L phosphate buffer at pH 7.4 and 0.1 µmol/L 1‐butyl‐3‐methyl imidazolium bromide (BMImBr) ionic liquid (IL) was used for CE measurements at 250 nm detection wavelength, 20 kV applied voltage and 25 °C. The rinsing protocol was significantly improved to reduce the adsorption of IL on the interior surface of capillary. Moreover, RPLC method was developed on α‐1‐acid glycoprotein (AGP) column. Mobile phase was 10 mmol/L phosphate buffer at pH 6.0 (100% v/v) and flow rate at 0.1 mL/min. As AGP is a chiral column, it was successfully separated l ‐MTX from its enantiomer impurity d ‐MTX. Good linearity of quantitative analysis was achieved with coefficients of determinations (r²) >0.995. The stability of drugs measurements was investigated with adequate recoveries up to 24 h storage time under ambient temperature. The limits of detection were <50 and 90 ng/mL by CE and RPLC, respectively. The using of short‐chain IL as an additive in BGE achieved 600‐fold sensitivity enhancement compared with conventional Capillary Zone Electrophoresis (CZE). Therefore, for the first time, the proposed methods were successfully applied to determine simultaneously the analytes in human plasma and urine samples at clinically relevant concentrations with fast and simple pretreatments. Developed IL‐assisted CE and RPLC methods were also applied to measure MTX levels in patients’ samples over time. Copyright © 2014 John Wiley & Sons, Ltd.     

Stable-isotope dilution analysis of galactose metabolites in human erythrocytes

An established gas chromatography/mass spectrometry (GC/MS) method, devised for stable-isotope dilution analysis of plasma galactose, was developed to allow determination of erythrocyte (red blood cell, RBC) concentrations of galactose-1-phosphate and other primary metabolites relevant in galactosaemia. Galactose-1-phosphate was enzymatically converted to galactose, and the aldononitrile pentaacetate derivative was separated by gas chromatography and determined by mass spectrometry using chemical ionisation and selected ion monitoring of the [MH-60](+) ion. U-(13)C-Labelled standard was used for quantification. Comparative measurements were conducted using established fluorimetric and radiometric enzymatic methods. The GC/MS analysis for galactose-1-phosphate was linear (range examined 0-600 micromol/L(RBC), packed cells), of acceptable repeatability at low and high concentrations (within and between run CVs <15%), with a limit of quantification of 0.01 micromol/L(RBC). With samples from patients with classical galactosaemia there was a linear correlation with conventional enzymatic assays (r(2) > 0.927). In erythrocytes from post-absorptive patients under treatment, Q188R-heterozygous parents, and healthy subjects, galactose-1-phosphate concentrations (mean +/- SD) were found to be 142 +/- 38 (n = 41), 1.4 +/- 0.2 (n = 8), and 1.9 +/- 0.5 (n = 33) micromol/L(RBC), respectively. In comparison, free galactose concentrations were 3.8 +/- 1.7, 0.49 +/- 0.19, and 0.43 +/- 0.20 mol/L(RBC), respectively. The procedure allowed simultaneous galactitol analysis and proved to be useful to trace incorporation of (13)C-label into erythrocyte galactose metabolites in a D-[1-(13)C]galactose in vivo turnover study.     

Determination of methotrexate, 7-hydroxymethotrexate,and 2,4-diamino-N10-methylpteroic acid by LC–MS/MS in plasma and cerebrospinal fluid and application in a pharmacokinetic analysis of high-dose methotrexate

A rapid and robust method for measuring methotrexate (MTX) and its two primary metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA), was developed for use in pharmacokinetic studies of plasma and cerebrospinal fluid samples collected from infants with malignant brain tumors. Sample aliquots (100?µL) were prepared for bioanalysis of MTX and metabolites using a Waters Oasis HLB microelution solid-phase extraction (SPE) plate. Chromatography was performed using a Phenomenex Synergi Polar-RP 4 µ 75?×?2.0?mm ID column heated to 40°C. A rapid gradient elution on a Shimadzu HPLC system was used, with mobile phase A consisting of water/formic acid (100/0.1?v/v) and mobile phase B consisting of acetonitrile/formic acid (100/0.1?v/v). Column eluent was analyzed using AB Sciex QTRAP 5500 instrumentation in electrospray ionization mode. The ion transitions (m/z) monitored were 455.2?→?308.1, 471.1?→?324.1, and 326.2?→?175.1 for MTX, 7-OHMTX, and DAMPA, respectively. The method was linear over 0.0022–5.5?µM for MTX, 0.0085–21?µM for 7-OHMTX, and 0.0031–7.7?µM for DAMPA. The method was applied to the analysis of serial plasma samples obtained from infants diagnosed with malignant brain tumors receiving high-dose methotrexate and results were compared to MTX concentrations from an immunoassay based on fluorescence polarization.     

Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

In this work, CE‐LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l ‐carnosine and l ‐alanyl‐glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE‐LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20–300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l ‐carnosine, l ‐alanyl‐glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.     

Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time‐of‐flight mass spectrometry

Linoleic acid (LA) and LA‐esters are the precursors of LA hydroperoxides, which are readily converted to 9‐ and 13‐hydroxy‐?octadecadienoic acid (HODE) and 9‐ and 13‐oxo‐?octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time‐of‐flight mass spectrometry (Q‐TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base‐hydrolyzed and followed by liquid–liquid extraction. Quantitative analyses were based on one‐point standard addition with isotope dilution. The Q‐TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted‐ion chromatograms were generated post‐acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7–35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9‐HODE, 13‐HODE, 9‐oxoODE and 13‐oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9‐HODE, 13‐HODE, 9‐oxoODE and 13‐oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one‐point standard addition. Target metabolites were simultaneously characterized based on the accurate Q‐TOFMS data. This is the first study of secondary LA metabolites using Q‐TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Phytochemical and In-Vivo Anti-Arthritic Significance of Aloe thraskii Baker in Combined Therapy with Methotrexate in Adjuvant-Induced Arthritis in Rats

Unlike other widely known Aloe species used for treatment of rheumatoid arthritis, this species suffers from a lack of sufficient studies on its biological and chemical characters. This is what drove us to perform this work to evaluate the in vivo anti-arthritic potential of its leaf ethanolic extract. The in vivo anti-arthritic activity of the leaf ethanolic extract at 100 and 200 mg/kg/day b.wt. was evaluated alone and in combination with methotrexate (MTX) using complete Freund’s adjuvant. Serum levels of rheumatoid factor, anti-cyclic citrullinated peptide (anti-CCP), cytokines pro-inflammatory marker, inflammatory mediator serum levels, and oxidative stress mediators were analyzed, in addition to liver function. Orientin, isoorientin, β-sitosterol, its palmitate and its glucoside were isolated. The combined therapy of MTX and the leaf ethanolic extract (especially at 200 mg/kg b.wt.) group showed better activity compared to MTX alone. Moreover, the combined therapy provided additional benefits in lowering the liver toxicity by comparison to MTX alone. We concluded that a synergetic combination of the leaf ethanolic extract and MTX is beneficial in the management of rheumatoid arthritis with fewer side effects on liver function, as well as the possibility of the leaf extract to stand alone as an effective natural anti-arthritic agent.     

Quantitative profiling of biomarkers related to B‐vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

Vitamins B₂ and B₆ serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large‐scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B₆ forms (pyridoxal 5′‐phosphate, pyridoxal, 4‐pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3‐hydroxykynurenine, xanthurenic acid and 3‐hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope‐labelled internal standards was added to 60 µL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable‐bond C8 column by a gradient‐type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75–123% and within‐day and between‐day coefficients of variance (CVs) were 2.5–9.5% and 5.4–16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B‐vitamin status and inflammation, and may prove useful in large‐scale epidemiological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

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.

Liquid chromatographic methods for the therapeutic drug monitoring of methotrexate as clinical decision support for personalized medicine: A brief review

Methotrexate (MTX) is an antifolate drug used for several diseases. Depending on the disease, MTX can be administered at low dose (LDMTX) in some autoimmune diseases, like rheumatoid arthritis, or at high dose (HDMTX) in some cancers, such as acute lymphoblastic leukemia. After absorption, MTX is metabolized in the liver to 7‐hydroxymethotrexate and in the intestine to 2,4‐diamino‐N10‐methylpteroic acid (DAMPA). Moreover, inside red blood cells, MTX is converted to active metabolites, MTX polyglutamates (MTXPGs), contributing to its pharmacodynamics. Owing to its narrow therapeutic range, and inter‐ and intra‐patient variability, either noneffectiveness and/or toxicity may occur. Because of the existence of a relationship between drug therapeutic outcome and its systemic concentration, therapeutic drug monitoring (TDM) may ensure the effectiveness and safety of MTX use. In order to monitor the optimization of patient clinical response profile, several analytical methods have been described for TDM in biological samples. These include liquid chromatography (LC) coupled with ultraviolet detection, fluorescence detection or mass spectrometry, each one presenting advantages and drawbacks. This paper reviews the most commonly used techniques for sample preparation and critically discusses the current LC methods applied for the TDM of MTX in biological samples, at LDMTX and HDMTX.     

On-line stacking capillary electrophoresis for analysis of methotrexate and its eight metabolites in whole blood

Therapeutic drug monitoring of methotrexate (MTX) and its metabolites is significant for the evaluation of treatment response of acute lymphoblastic leukemia and the prevention of adverse effects. Those eight metabolites including 7-hydroxymethotrexate, MTX-polyglutamate derivatives (MTX-(Glu)(n), n=2-7), and 2,4-diamino-N(10)-methylpteroic acid, especially the MTX-(Glu)(n), only exist in blood cells and also present antifolate effects. Therefore, whole blood analysis has importance in clinical therapy. This study combined solid-phase extraction and on-line stacking capillary electrophoresis to examine the levels of MTX and its eight metabolites in whole blood. A higher conductivity buffer (HCB) was used to accumulate large amount of samples into a narrow zone, which resulted in effective stacking and sharp peaks. A fused-silica capillary was filled with phosphate buffer (80 mM, pH 2.5) containing 15% v/v tetrahydrofuran and 100 mM sodium dodecyl sulfate. Then HCB (100 mM phosphate, pH 2.5; 1 psi, 60 s) was injected for accumulating large volume of analytes (1 psi, 200 s). Owing to the pH difference between sample zone and HCB, dynamic pH junction was generated for focusing. Meanwhile, sweeping made further stacking by using sodium dodecyl sulfate in phosphate buffer. The limits of detection (S/N=3) were 0.1 microM for MTX, 0.2 microM for 7-hydroxymethotrexate and 2,4-diamino-N(10)-methylpteroic acid, 0.3 microM for MTX-(Glu)(n=2--5), and 0.5 microM for MTX-(Glu)(n=6--7). During validation, this method showed good linearity (r> / =0.9914) and reproducibility (relative standard deviation and relative error, both less than 13%). The applications were performed to monitor blood MTX and its metabolites in acute lymphoblastic leukemia patients. The blood concentrations could provide some information related to therapeutic response and adverse effects.     

Identification and quantification of the antipsychotics risperidone,aripiprazole, pipamperone and their major metabolites in plasma using ultra‐high performance liquid chromatography–mass spectrometry

The antipsychotics risperidone, aripiprazole and pipamperone are frequently prescribed for the treatment in children with autism. The aim of this study was to validate an ultra‐high performance liquid chromatography–mass spectrometry method for the quantification of these antipsychotics in plasma. An ultra‐high performance liquid chromatography–mass spectrometry assay was developed for the determination of the drugs and metabolites. Gradient elution was performed on a reversed‐phase column with a mobile phase consisting of ammonium acetate, formic acid in methanol or in Milli‐Q ultrapure water at a flow rate of 0.5 mL/min. The method was validated according to the US Food and Drug Administration guidelines. The analytes were found to be stable enough after reconstitution and injection of only 5 μL improved the accuracy and precision in combination with the internal standard. Calibration curves of all five analytes were linear. All analytes were stable for at least 72 h in the autosampler and the high quality control of 9‐OH‐risperidone was stable for 48 h. The method allows quantification of all analytes. The advantage of this method is the combination of a minimal injection volume, a short run‐time, an easy sample preparation method and the ability to quantify all analytes in one run. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of thyroid hormones and their metabolites in tissue using SPE UPLC-tandem MS

A solid‐phase liquid chromatography tandem mass spectrometry (SPE LC‐MS/MS) method was developed to determine thyroid hormones and their metabolites in tissue samples. The separation was achieved using reversed‐phase ultra‐performance liquid chromatography (UPLC); the mass spectrometric detection was achieved by positive electrospray ionization and multiple reaction monitoring. Prior to the UPLC separation a sample cleanup with a cation exchange was performed. ¹³ C₆ labeled internal standards were used for the thyroid hormones and their metabolites. The method was linear over a range from 0.23 to 90 nmol/L for thyroxine and from 0.23 to 9 nmol/L for the metabolites. The lower limit of quantification ranged from 0.98 to 1.73 pg on column. Intra‐ and total assay variation were <10 and <15%, respectively. This method enables us to link thyroid hormone tissue concentrations to local iodothyronine deiodinase expressions, which will enhance our understanding of the regulation of thyroid hormone metabolism on the tissue level. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of nanofiber‐packed SPE for determination of salivary‐free cortisol using fluorescence precolumn derivatization and HPLC detection

Salivary cortisol has emerged as an easy‐to‐collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary‐free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high‐throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro‐column was designed. The LOD of cortisol was 0.01 μg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 μg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 μg/L. The nanofiber‐packed SPE overcame the low extraction recovery and bad clean‐up effect of the conventional methods, and increased the sensitivity and selectivity of the method.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography

An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH₂PO₄) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L⁻¹ to 5 mg L⁻¹ for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 μg L⁻¹, and the relative standard deviations (RSD, n = 7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.     

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

Methotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250?×?4.6?mm, 5?µm) column, methanol, and 0.05% trifloroacetic acid (36:64?v/v) as mobile phase with a flow rate of 1?mL/min, detection wavelength of 290?nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0?µg/mL for methotrexate and 0.030–1.0?µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.

The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.