Development and validation of an LC-MS/MS method for simultaneous quantification of voriconazole and its main metabolite voriconazole N-oxide in human plasma and its clinical application

An LC-MS/MS method was developed for simultaneous determination of voriconazole and its main metabolite voriconazole N-oxide in human plasma. Plasma pretreatment was simple one-step protein precipitation with acetonitrile, and separation was achieved on a Hypersil GOLD aQ column. A triple-quadrupole tandem mass spectrometer with electrospray ionization source was operated by multiple reaction monitoring (MRM) in positive ion mode, and the precursor–product ion pairs used for MRM were m/z 350.1→281.0, 365.8→224.1, and 531.3→489.2 for voriconazole, voriconazole N-oxide, and internal standard, respectively. The total analytical run time was 6?min. The proposed method was linear over the range 0.001–1?µg/mL for voriconazole and voriconazole N-oxide. The lower limit of quantification was 1?ng/mL for both of them. The specificity, accuracy, precision, recovery, matrix effect, stability as well as dilution integrity of this method were within acceptable limits during validation period. This method was successfully applied to therapeutic drug monitoring of voriconazole and to evaluate its metabolite profile.     

A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study

A new, rapid, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous quantification of tenofovir and lamivudine in human plasma using abacavir as an internal standard. An API‐4000 LC‐MS/MS with electrospray ionization was operated in multiple‐reaction monitoring mode for the analysis. The analytes were extracted from plasma by solid‐phase extraction technique using an Oasis HLB cartridge. The reconstituted samples were chromatographed on a Chromolith ROD speed C₁₈ column using a mixture of 0.1% formic acid in water and acetonitrile (90:10 v/v) at a flow‐rate of 1 mL/min. The method was validated as per the FDA guidelines. The calibration curves were found to be linear in the range of 5–600 ng/mL for tenofovir and 25– 4000 ng/mL for lamivudine. The intra‐ and inter‐day precision and accuracy results were well within the acceptable limits. A run time of 2.8 min consumed for each sample made it possible to analyze more samples per day. The proposed assay method was found to be applicable to a pharmacokinetic study in human male volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Validation and application of an LC-MS/MS method for the simultaneous quantification of 13 pharmaceuticals in seawater

Knowledge of the presence of micropollutants such as pharmaceuticals, in coastal areas, is very limited; therefore, the main objective of this study was to optimize and validate a new analytical method for the quantitative analysis of 13 multiclass pharmaceuticals in seawater. Target compounds included antibiotics, non-steroidal anti-inflammatory drugs, β-blockers, lipid regulators and one psychiatric drug. A combination of solid-phase extraction and liquid chromatography coupled with multiple mass spectrometry enabled their detection at the low nanogram per litre level. The limits of quantification varied between 1 and 50 ng L^-1, for most components the linearities were more than 0.99 and the recoveries obtained in seawater (95–108%) were satisfactory. This method was applied to seawater and estuarine water samples collected in the Belgian coastal zone, to assess the prevalence of common pharmaceuticals in this marine environment. Seven pharmaceuticals, including compounds of which the presence in marine environments had not been reported earlier, were detected, with salicylic acid and carbamazepine being the most abundant, in concentrations up to 855 ng L^-1.     

Development and validation of a specific and sensitive LC-MS/MS method for quantification of urinary catecholamines and application in biological variation studies

Catecholamines are a class of biogenic amines that play an important role as neurotransmitters and hormones. We developed and validated a rapid, specific and sensitive LC-MS/MS method for quantitative determination of catecholamines in human urine. Linearity, specificity, sensitivity, precision, accuracy, matrix effect, carryover, analyte stability, method comparison and reference range were evaluated. The catecholamine measurements were not affected by 35 structurally-related drugs and metabolites. The outstanding specificity was achieved by use of a specific diphenylborate-based solid phase extraction and subsequent selective LC-MS/MS analysis. Excellent sensitivity, accuracy and precision (average intra-assay variations <2.9 % and inter-assay variations <4.6 %) were obtained. The method was successfully applied in the study of day-to-day biological within- and between-subject variations of 25 healthy people under free-living conditions over three consecutive days. We observed that catecholamine excretions for second morning sampling had least day-to-day within-subject variation and excellent reproducibility. This work is one of the rare studies on these topics and represents the first utilization of advanced LC-MS/MS technology. Additionally, we found significant correlations between spot and conventional 24 h collections of human urine (n?=?22, r?>?0.853, p?LC-MS/MS method presented here is rapid, sensitive and specific, which could be an advantage in clinical laboratories. Graphical Abstract

Diurnal variation of urinary catecholamines for 25 healthy people in three consecutive days     

Development and validation of an SPE-GC-MS/MS taste and odour method for analysis in surface water

The goal of this research was to develop a robust method for taste and odour compounds that can be implemented by laboratories with mass spectrometers lacking chemical ionisation capabilities or specialised sample introduction hardware that are commonly used for taste and odour methods. Development, optimisation, and validation of a solid-phase extraction method using liquid injection and gas chromatography – tandem mass spectrometry detection with electron impact ionisation are described. Camphor was used as an internal standard, and through method development and robustness testing it was shown to extract similarly to other taste and odour compounds, making it a cost-effective alternative to deuterated analogs. The instrumental parameters and extraction procedure were fully optimised prior to assessing the method’s linearity, precision, and accuracy. Using a 2000-fold enrichment factor, method recoveries for priority compounds geosmin (GSM) and 2-methylisoborneol (2-MIB) were >90%. Excellent linearity was obtained from the reportable detection limits up to 200 ng L^?1 and precision %relative standard deviations were 8.5% and 10.9% for GSM and 2-MIB, respectively. Detection limits of 0.9 and 5.5 ng L^?1 for GSM and 2-MIB respectively were deemed fit-for-purpose in comparison to their odour thresholds. Validation data were also obtained for other commonly analysed taste and odour compounds, including 2,4,6-trichloroanisole, 2-isopropyl-3-methoxypyrazine, and 2-isobutyl-3-methoxypyrazine. The validated method was used to screen surface waters in Nova Scotia, Canada for presence of taste and odour compounds, highlighting the presence of GSM on the east coast of Canada.     

Development and validation of an analytical method by LC-MS/MS for the quantification of estrogens in sewage sludge

A sensitive method for the simultaneous analysis of five estrogens in sewage sludge was developed. The extraction and purification steps were optimized and the matrix effects were evaluated. The chromatographic gradient was optimized to limit matrix effects and the analysis step was performed by LC-MS/MS. The method consists of an ASE® extraction with a solvent mixture water/methanol 80/20?v/v at 100?°C followed by two consecutive purifications on Oasis HLB® and florisil cartridges. A thorough validation of the developed method was performed. Recoveries determined at two different spiking levels ranged between 86% and 126% depending on the molecule. Repeatability was evaluated on five replicates of the same sludge sample spiked at two different levels and measuring native estrogens in triplicates of 12 sludge samples. Relative standard deviations obtained a range of between 2% and 27%. Reproducibility was also studied by analyzing the same sludge on four different days: the relative standard deviation ranged between 14% and 20% for E1, βE2 and E3. For αE2, poor reproducibility (68%) was observed but it was linked to the very low quantity of αE2 present in the sludge sample and not to the method performance. The specificity of the method was evaluated on various sludge samples spiked at different spiking levels showing that performances of the proposed method were not modified by matrix effects. Finally, sensitivity of the method was evaluated taking into account both instrumental sensitivity and matrices; the estimated limits of quantification were around 1 ng/g for E1, between 2 and 4 ng/g for αE2, βE2, and E3 and around 5 ng/g for EE2.     

Development of a capillary electrophoresis method for the simultaneous analysis of artificial sweeteners, preservatives and colours in soft drinks

A rapid capillary electrophoresis method was developed simultaneously to determine artificial sweeteners, preservatives and colours used as additives in carbonated soft drinks. Resolution between all additives occurring together in soft drinks was successfully achieved within a 15-min run-time by employing the micellar electrokinetic chromatography mode with a 20 mM carbonate buffer at pH 9.5 as the aqueous phase and 62 mM sodium dodecyl sulfate as the micellar phase. By using a diode-array detector to monitor the UV-visible range (190-600 nm), the identity of sample components, suggested by migration time, could be confirmed by spectral matching relative to standards.     

Development and validation of LC-MS/MS method for the quantification of oxcarbazepine in human plasma using an experimental design

A rapid tandem mass spectrometric (MS-MS) method for the quantification of Oxcarbazepine (OXB) in human plasma using imipramine as an internal standard (IS) has been developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 3.0 min, using a mobile phase of acetonitrile-10 mM ammonium formate (90 : 10 v/v) at a flow rate of 0.3 ml/min. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 253>208 and m/z 281>86 for OXB and the IS respectively. Calibration curves were linear over the concentration range of 0.2-16 mug/ml (r>0.999) with a limit of quantification of 0.2 mug/ml. Analytical recoveries of OXB from spiked human plasma were in the range of 74.9 to 76.3%. Plackett-Burman design was applied for screening of chromatographic and mass spectrometric factors; factorial design was applied for optimization of essential factors for the robustness study. A linear model was postulated and a 2(3) full factorial design was employed to estimate the model coefficients for intermediate precision. More specifically, experimental design helps the researcher to verify if changes in factor values produce a statistically significant variation of the observed response. The strategy is most effective if statistical design is used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic and bioequivalence studies.     

Development of a solvent-free method for the simultaneous identification/quantification of drugs of abuse and their metabolites in environmental water by LC-MS/MS

This work details a rapid analytical method using direct sample injection for the simultaneous identification/quantification of 22 drugs of abuse, including some of their major metabolites, in environmental samples. This has been developed using a hybrid triple quadrupole-linear ion trap-mass spectrometer (QqLIT). With the increasing sensitivity of today's tandem mass spectrometers, direct injection analysis of water samples has become an attractive alternative to traditional analytical protocols, which often include a preliminary pre-concentration step. What's more, this kind of analysis is in accordance with many of the main objectives of so-called green analytical chemistry, or environmentally friendly practice. The analytical performance of the LC-MS/MS method was evaluated in three different water matrices (surface water, influent and effluent wastewater). Data acquisition was carried out in selected reaction monitoring (SRM) mode under time-scheduled conditions, monitoring two SRM transitions for simultaneous identification/quantification of all target compounds in the samples. Additionally, an experiment was performed using the information-dependent acquisition (IDA) scan to carry out the identification of those analytes for which the second transition was present at a low intensity. Finally, the two methodologies developed were applied to real samples for evaluation.     

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues

A highly sensitive and specific LC‐MS/MS method was developed for simultaneous estimation of acetyl co‐enzyme A (ACoA) and malonyl co‐enzyme A (MCoA) in surrogate matrix using n‐propionyl co‐enzyme A as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Simple acidification followed by dilution using an assay buffer process was used to extract ACoA, MCoA and IS from surrogate matrix and tissue samples. The total run time was 3 min and the elution of both analytes (ACoA, MCoA) and IS occurred at 1.28 min; this was achieved with a mobile phase consisting of 5 mM ammonium formate (pH 7.5)–acetonitrile (30:70, v/v) delivered at a flow rate of 1 mL/min on a monolithic RP‐18e column. A linear response function was established for the range of concentrations 1.09–2187 and 1.09–2193 ng/mL for ACoA and MCoA, respectively. The intra‐ and inter‐day precision values for ACoA and MCoA met the acceptance as per FDA guidelines. ACoA and MCoA were stable in a battery of stability studies viz. bench‐top, auto‐sampler and long‐term. The developed assay was used to quantitate ACoA and MCoA levels in various tissues of rat. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays

In the present study, a rapid and sensitive LC-ESI-MS/MS method for quantification of (S)-fluoxetine as a native marker in mass spectrometry (MS) binding assays addressing the human serotonin transporter (hSERT) was developed and validated. The concept of MS binding assays based on mass spectrometric quantification of a nonlabeled marker recently introduced by us represents a promising alternative to conventional radioligand binding without the drawbacks inherently connected with radioisotope labeling. For high-performance liquid chromatography (HPLC), a 20 × 2-mm RP-18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L⁻¹, pH 9.5) at a ratio of 80:20 (v/v) and a flow rate of 800 μL min⁻¹ in an isocratic mode were used, resulting in a chromatographic cycle time of 60 s. Employing [²H₅]fluoxetine as internal standard enabled ESI-MS/MS quantification of (S)-fluoxetine between 3 nmol L⁻¹ and 50 pmol L⁻¹ (LLOQ) in matrix obtained from binding experiments without the need of any sample preparation. Validation of the method showed that linearity, intra-, and inter-batch accuracy as well as precision meet the requirements of the FDA guidance for bioanalytical method validation. Considering sensitivity and speed, the established method is clearly superior to those published for biological matrices so far. Furthermore, the method was transferred to other RP-18 columns of different lengths and respective validation experiments demonstrated its versatility and chromatographic robustness. Finally, the newly developed method was successfully applied to MS binding assays for hSERT. The affinity determined for (S)-fluoxetine in saturation experiments was in good agreement with literature data obtained in respective radioligand binding assays.     

Development and validation of a reliable and rapid LC‐MS/MS method for simultaneous quantification of sacubitril and valsartan in rat plasma and its application to a pharmacokinetic study

A selective, sensitive and rapid liquid chromatographic method with electrospray ionization tandem mass spectrometric detection has been developed and validated for simultaneous quantification of sacubitril and valsartan in rat plasma using telmisartan as internal standard (IS). The analytes were extracted by deprotenization of 50 μL of plasma sample using 200 μL of acetonitrile. In a short chromatographic run of 1.50 min run time, separation was achieved on a Hypersil Gold C₁₈ column using a mobile phase composed of 0.1% formic acid in Milli‐Q water–0.1% formic acid in acetonitrile in gradient elution mode. The quantification of target compounds was performed in a positive electrospray ionization mode and multiple reaction monitoring. Response was a linear function of concentration in the ranges of 0.5–20,000 ng/mL for both analytes, with r² > 0.9997. The intra‐ and inter‐day precision and accuracy results were <15% and acceptable as per US Food and Drug Administration guidelines. Stability of compounds were established in a battery of stability studies, i.e. bench‐top, autosampler and long‐term storage stability as well as freeze–thaw cycles. The validated method can be used as a routine method to support pharmacokinetic studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of ethionamide and ethionamide sulfoxide in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous quantification of ethionamide and ethionamide sulfoxide in human plasma (300 µL) using prothionamide as an internal standard (IS). Solid‐phase extraction was used to extract ethionamide, ethionamide sulfoxide and IS from human plasma. The chromatographic separation of ethionamide, ethionamide sulfoxide and IS was achieved with a mobile phase consisting of 0.1% acetic acid : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Peerless Basic C₁₈ column. The total run time was 3.5 min and the elution of ethionamide, ethionamide sulfoxide and IS occurred at 2.50, 2.18 and 2.68 min, respectively. A linear response function was established for the range of concentrations 25.7–6120 ng/mL (r > 0.998) for ethionamide and 50.5–3030 ng/mL (r > 0.998) for ethionamide sulfoxide. The intra‐ and inter‐day precision values for ethionamide and ethionamide sulfoxide met the acceptance as per FDA guidelines. Ethionamide and ethionamide sulfoxide were stable in battery of stability studies, viz. bench‐top, autosampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

Bioanalytical method development and validation of novel antithrombotic agent S002-333 by LC-MS/MS and its application to pharmacokinetic studies

A rapid, sensitive and simple liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method using an electrospray ionization (ECI) source for the quantification of novel anti‐thrombotic agent S002‐333 [2‐(4‐methoxy‐benzenesulfonyl)‐2,3,4,9‐tetrahydro‐1H‐β‐carboxylic acid amide] in rabbit plasma was developed and validated. The extraction from plasma was carried out by simple protein precipitation extraction method. The chromatographic separation was performed on an Ultramex Cyno, (150 × 4.6 mm, 5 µm) with a guard column, using acetonitrile–water (75:25,v/v) with flow rate of 0.6 mL/min as the mobile phase. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 386.4/215.4 for S002‐333 and m/z 393.4/171for the internal standard dexamethasone, using positive ion mode. The MS/MS response was linear over the concentration range from 1.56 to 200 ng/mL, with a lower limit of detection of 0.78 ng/mL. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations and showed no significant matrix effect. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies Copyright © 2010 John Wiley & Sons, Ltd.     

A sensitive UHPLC–MS/MS method for the simultaneous quantification of three lignans in human plasma and its application to a pharmacokinetic study

The aim of this study was to develop an analytical method to simultaneously analyze schizandrin, schizandrol B, and gomisin N lignans in human plasma using ultra high performance liquid chromatography with tandem mass spectrometry. The three lignans were separated using a mobile phase of water and acetonitrile containing 0.02% acetic acid equipped with a Kinetex C₁₈ column (2.1 mm × 50 mm, 1.7 μm). This analysis was achieved by multiple reaction monitoring mode in an electrospray interface. The mass transitions were m /z 433.1→384.0 for schizandrin, 398.8→367.8 for schizandrol B, and 400.6→299.8 for gomisin N. Liquid–liquid extraction with methyl tert‐butyl ether was used to obtain the three lignans. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. The calibration curves for the three lignans in human plasma were 0.05–50 ng/mL and displayed excellent linearity with correlation coefficients greater than 0.99. Precision for all three lignans was within 11.23%. The accuracy was 88.3–99.0% for schizandrin, 90.6–103.4% for schizandrol B, and 90.2–103.5% for gomisin N. The developed simultaneous analytical method satisfied the criteria of international guidance and could be successfully applied to the pharmacokinetic study of three lignans after oral administration of Schisandrae Fructus extract powder to humans.     

Multiresidue fully automated online SPE-HPLC-MS/MS method for the quantification of endocrine-disrupting and pharmaceutical compounds at trace level in surface water

The present work describes the development and validation of a sensitive method for the determination of traces of diverse groups of pharmaceuticals and endocrine disruptors in surface water. Thirty-seven substances have been selected, including 10 pesticides, 6 hormonal steroids and assimilates, 12 pharmaceuticals, 5 alkylphenols, 1 chlorophenol and 3 other well-known human contaminants, 1 UV filter and 2 plasticisers. An automated online solid-phase extraction (SPE) is directly coupled to liquid chromatography–tandem mass spectrometry. Different SPE columns have been tested, and the injection volume has been optimised. The developed analytical methodology is based on the direct injection of 2.5 mL of water sample acidified at pH 1.6 on an Oasis HLB loading column (20 × 2.1 mm) with 5-µm particles. Then, the chromatographic separation is achieved on a Kinetex XB C18 (100 × 2.1 mm; 1.7 µm) column, and the quantification is realised in multiple-reaction monitoring mode. The online SPE step warrants minimal sample handling, low solvent consumption, high sample throughput, saving time and costs. This method allows the quantification of the target analytes in the lower ng/L concentration range, with limits of quantification (LQs) between 100 pg/L and 10 ng/L, 26 compounds having LQ lower than 1 ng/L. The monitoring of two selected MS/MS transitions for each compound allows the reliable confirmation of positive findings even at the LQ level. The developed and validated methodology has been applied to the analysis of various real samples from two French rivers. Twelve target compounds have been detected in the environmental samples, and the major pollutants are pharmaceuticals usually used by humans (paracetamol, carbamazepine, oxazepam, ketoprofen, trimethoprim). The pesticides atrazine and carbendazim have been ubiquitously detected in real samples too. Metronidazole, sulfamethoxazole and diuron were also frequently quantified in the water samples.