Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5%?v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS2 results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2?μg/mL (extracts).     

Forensic confirmatory analysis of ethyl sulfate—A new marker for alcohol consumption—by liquid-chromatography/electrospray ionization/tandem mass spectrometry

Ethyl sulfate (EtS)--a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others--was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M - H]- m/z 125 and product ions m/z 97 [HSO4]- and m/z 80 [SO3]-. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M - H]- m/z 130, product ions m/z 98 [DSO4]- and m/z 80 [SO3]-). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.     

Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulfate in urine by liquid chromatography/electrospray tandem mass spectrometry

A liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) method for the determination in urine samples of two ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), was developed and validated. Pentadeuterated EtG was used as internal standard for both EtG and EtS. In addition to the surviving ions, two MS/MS reactions were monitored for each analyte, with the deprotonated molecule as precursor ion: m/z 221 --> 75, m/z 221 --> 85 (EtG), and m/z 125 --> 97, m/z 125 --> 80 (EtS). Sample pretreatment, though very simple and rapid (1:50 water dilution and centrifugation of 50 muL of urine), was found to contain the occurrence of matrix effects. The method was accurate and precise over the linear dynamic range (0.05-10 mg/L). The analytes were stable in frozen urine for at least 1 month. The assay was applied to several authentic urine samples from social drinkers and to alcoholic beverages.     

Highly sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by solid‐phase extraction and liquid chromatography‐tandem mass spectrometry

Using bamboo‐activated charcoal as SPE adsorbent, a novel SPE method was developed for the sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by rapid‐resolution LC‐ESI‐MS/MS. Important parameters influencing extraction efficiency, including type of eluent, eluent volume, sample pH, volume and flow rate, were investigated and optimized. Under the optimal extraction conditions (eluent: 8 mL methanol, pH: 7; flow rate: 4 mL/min; sample volume: 100 mL), low LODs (0.01–0.02 ng/mL), good repeatability (6.2–8.3%) and wide linearity range (0.10–10 ng/mL) were obtained. Satisfied results were achieved when the proposed method was applied to determine the two target compounds in real‐world environmental water samples with spiked recoveries over the range of 80.5–119.8%. All these facts indicate that trace determination of tetrabromobisphenol A and bisphenol A in real‐world environmental water samples can be realized by bamboo‐activated charcoal SPE‐rapid resolution‐LC‐ESI‐MS/MS.     

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC–ESI–MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.     

A high-performance liquid chromatographic-tandem mass spectrometric method for the determination of ethyl glucuronide and ethyl sulfate in urine validated according to forensic guidelines

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are powerful markers for alcohol intake and abuse. Several analytical procedures for the quantification of EtG and EtG in serum and urine have been developed so far. Many of the published methods show limits of detections (LODs) or limits of quantifications (LOQs) for EtG in urine within the range of 0.1 mg/L or higher. Since this is the actual cutoff value for proving abstinence in Germany, problems may occur if urine samples are highly diluted. In this paper, the validation of a highly sensitive, fast and simple LC-MS-MS for the determination of EtG and EtS in urine is described. The calibration curves for EtG and EtS is linear over the whole range (0.025-2.0 mg/L). Very low detection limits can be achieved (LOD: EtG 0.005 mg/L, EtS 0.005 mg/L; and LOQ: EtG 0.019 mg/L, EtS 0.015 mg/L). All data for selectivity, precision and accuracy, recovery, as well as for the processed sample and the freeze/thaw stability, comply with the guidelines of the German Society of Toxicological and Forensic Chemistry. Strong matrix-related effects can be compensated for by using an internal standard. Finally, the applicability of the procedure is proven by analysis of 87 human urine samples and by successful participation in interlaboratory comparison tests.     

Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters,ethyl glucuronide,and ethyl sulfate followed by LC-MS/MS quantification

Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25–50 ng/g, with calibration curves to 2,500–5,000 ng/g. EtG and EtS linear dynamic ranges were 5–1,000 and 2.5–500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2–96.5 %. Matrix effects ranged from ?84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (?20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption.

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection (Part II)

A direct, versatile method for the determination of ethyl glucuronide (EtG), a biomarker of ethanol consumption, in urine has been developed using reversed-phase liquid chromatography with pulsed electrochemical detection (PED). EtG and methyl glucuronide (MetG), which serves as an internal standard, are readily separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH allows for the detection of all glucuronides using PED at a gold working electrode. Upon optimization, EtG was found to have a limit of detection of 0.03 μg/mL (7 pmol; 50 μL injection volume) and repeatability at the limit of quantitation of 1.7%R.S.D. (relative standard deviation). Solid-phase extraction (SPE) using an aminopropyl phase was used to remove interferents in urine samples prior to their analysis. Compound recovery following SPE was approximately 50 ± 2%. The forensic utility of this method was further validated by the analysis of 29 post-mortem urine specimens, whose results agreed strongly with certified determinations.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Rapid and simple determination of psychotropic phenylalkylamine derivatives in urine by direct injection liquid chromatography–electrospray ionization–tandem mass spectrometry

A direct injection liquid chromatography–electrospray ionization–tandem mass spectrometric method (LC‐ESI‐MS/MS) was developed and validated for the rapid and simple determination of 13 phenylalkylamine derivatives. Eight deuterium‐labeled compounds were prepared for use as internal standards (ISs) to quantify the analytes. Urine samples mixed with ISs were centrifuged, filtered through 0.22 µm filters and then injected directly into the LC‐ESI‐MS/MS system. The mobile phase was composed of 0.2% formic acid and 2 mM ammonium formate in distilled water and 0.2% formic acid and 2 mM ammonium formate in acetonitrile. The analytical column was a Capcell Pak MG‐II C₁₈ (150 × 2.0 mm i.d., 5 µm, Shiseido). Separation and detection of the analytes were accomplished within 10 min. The linear ranges were 5–750 ng/mL (ephedrine and fenfluramine), 10–750 ng/mL (3,4‐methylenedioxyamphetamine, phendimetrazine, methamphetamine, 3,4‐methylenedioxyethylamphetamine and benzphetamine), 20–750 ng/mL (norephedrine, amphetamine, phentermine and ketamine) and 30–1000 ng/mL (3,4‐methylenedioxymethamphetamine and norketamine), with determination coefficients, R², ≥ 0.9967. The intra‐day and inter‐day precisions were within 19.1%. The intra‐day and inter‐day accuracies ranged from ?16.0 to 18.7%. The lower limits of quantification for all the analytes were lower than 26.5 ng/mL. The applicability of the method was examined by analyzing urine samples from drug abusers (n = 30). Copyright © 2012 John Wiley & Sons, Ltd.     

On‐line solid‐phase extraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of trace tributyltin and triphenyltin in water samples

On‐line solid‐phase extraction (SPE) for pre‐concentration and sample cleanup is one strategy to reduce matrix effects and to simultaneously improve detection sensitivity in liquid chromatography/mass spectrometry (LC/MS). This paper describes an on‐line SPE‐LC/MS method for the determination of tributyltin (TBT) and triphenyltin (TPhT) at trace levels in water samples. The direct coupling of an on‐line C₁₈ pre‐column to LC/MS was used to pre‐concentrate TBT and TPhT at trace levels from waters and to remove interfering matrix effects. Pre‐concentration was followed by separation of TBT and TPhT on a C₁₈ column using a mobile phase containing 0.1% (v/v) HCOOH/5 mM HCOONH₄ and methanol. While both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) can be interfaced with MS for the detection of TBT and TPhT, ESI‐MS was preferred for this application. The calibration curve for the targets was linear in the concentration range 0.1–30 µg L^?1. The detection limit (signal‐to‐noise (S/N) ratio = 3) was 0.02 µg L^?1 when 3.0 mL of sample was enriched on the C₁₈ pre‐column. The recoveries of TBT and TPhT in spiked waters were from 81.0 to 101.9%. The reproducibilities for the analysis of the standard mixture (10 µg L^?1) for TBT and TPhT were 13.1 and 5.0%, respectively. The developed method was an easy and fast way to analyze TBT and TPhT in water samples. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library search

Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the ‘screen’ and ‘confirmation’ goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI‐MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid‐liquid and solid‐phase extraction protocols were coupled to LC/ESI‐MS/MS using a 1.8‐µm particle size analytical column operated at 50°C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive‐ion ESI‐MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1–10 µg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens – based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two‐step (immunoassay and GC/MS) approach in the past. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of stable‐isotope dilution liquid chromatography–tandem mass spectrometric method for determination of salivary progesterone

A method for the quantification of progesterone (PROG) in human saliva using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata™‐X cartridge, and subjected to LC‐ESI‐MS/MS. Quantification was based on selected reaction monitoring, and deuterated PROG was used as the internal standard. This method allowed the reproducible (intra‐ and inter‐assay relative standard deviations, <2.2%) and accurate (analytical recovery, 96.6–99.7%) quantification of the salivary PROG using a 400 μL sample, and the limit of quantification was 12.5 pg/mL. The developed method enabled detection of the variation in the salivary PROG concentrations of healthy volunteers during the menstrual cycle and measurement of the salivary concentrations of pregnant women. The method is expected to be an alternative to the blood PROG monitoring in clinical examinations, because saliva collection is easy, non‐invasive and repeatable. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid

The purpose of the present work was to evaluate the synergistic effect of ionization type, sample preparation technique, and bio-fluid on the presence of matrix effect in quantitative liquid chromatography (LC)-MS/MS analysis of illicit drugs by post-column infusion experiments with morphine (10-microg/mL solution). Three bio-fluids (urine, oral fluid, and plasma) were pretreated with four sample preparation procedures [direct injection, dilution, protein precipitation, solid-phase extraction (SPE)] and analyzed by both LC-electrospray ionization (ESI)-MS/MS and LC-atmospheric pressure chemical ionization (APCI)-MS/MS. Our results indicated that both ionization types showed matrix effect, but ESI was more susceptible than APCI. Sample preparation could reduce (clean up) or magnify (pre-concentrate) matrix effect. Residual matrix components were specific to each bio-fluid and interfered at different time points in the chromatogram. We evaluated matrix effect in an early stage of method development and combined optimal ionization type and sample preparation technique for each bio-fluid. Simple dilution of urine was sufficient to allow for the analysis of the analytes of interest by LC-APCI-MS/MS. Acetonitrile protein precipitation provided both sample clean up and concentration for oral fluid analysis, while SPE was necessary for extensive clean up of plasma prior to LC-APCI-MS/MS.     

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.     

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.