An ultra-sensitive online SPE-LC-MS/MS method for the quantification of levonorgestrel released from intrauterine devices

A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (d-(−)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.     

Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS

A sensitive LC-MS/MS assay for quantification of total and free concentrations of R- and S-warfarin in plasma was required to support clinical studies on warfarin enantiomers. Several ultrafiltration devices were evaluated for separation of free warfarin from plasma proteins. The highest precision and lowest non-specific binding was obtained for Centrifree ultrafiltration devices. R- and S-warfarin were extracted from plasma (total) and ultrafiltrate (free) by liquid–liquid extraction with methyl tert-butyl ether using d₆-warfarin as internal standard. Mean extraction recovery was 68 ± 4%. The enantiomers were separated on a Chirobiotic V column with isocratic elution using 40% methanol and 0.03% acetic acid in water. Negative mode electrospray ionisation was used for MS/MS detection, monitoring the ion transition m/z 307/161. Calibration curves (quadratic, weighted 1/x) were fitted over the range of 20–2,000 ng/ml (r ² ≥ 0.995) in plasma and 0.5–20 ng/ml (r ² ≥ 0.998) in ultrafiltrate. The lower limit of quantification for R- and S-warfarin was 0.5 ng/ml in ultrafiltrate. Intra- and interday precision (% RSD) and bias were within 10% in all cases, and matrix effects were negligible. The assay was applied successfully to analysis of samples from clinical studies.     

Simultaneous quantitative analysis of letrozole, its carbinol metabolite, and carbinol glucuronide in human plasma by LC-MS/MS

Letrozole is an efficient endocrine treatment of postmenopausal breast cancer, however, not all patients benefit from this treatment, and moreover, severe side-effects like arthralgia frequently lead to discontinuation. To better understand inter-individual variability in drug response and side-effects, plasma analysis of steady-state concentrations of letrozole and its major metabolites is crucial. We developed a rapid, sensitive, and specific method for the simultaneous quantification of letrozole and its metabolites 4,4′-(hydroxymethylene)dibenzonitrile (carbinol) and bis(4-cyanophenyl)methyl hexopyranosiduronic acid (carbinol-gluc) by UHPLC-ESI-MS/MS using in-house synthesized, stable isotope-labeled internal standards. Following solid-phase extraction in BondElut C18 96-well plates, the analytes were separated on a ZORBAX Eclipse XDB-C18 column (1.8 μm, 4.6 × 50 mm) with a gradient of acetonitrile in 0.1% acetic acid in water and detected on a triple quadrupole mass spectrometer with electrospray ionization in the multiple reaction monitoring mode. Lower limits of quantification were 20, 0.2, and 2 nM for letrozole, carbinol, and carbinol-gluc, respectively. The assay has been validated according to FDA guidance and applied to the analysis of 20 plasma samples of postmenopausal breast cancer patients treated with 2.5 mg of letrozole per day. Mean plasma levels (±SD) were 366 ± 173, 0.38 ± 0.09, and 34 ± 12 nM for letrozole, carbinol, and carbinol-gluc, respectively. Our rapid and sensitive mass spectrometry based method enables future pharmacokinetic investigations of letrozole outcome.     

Quantitative determination of Phakellistatin 13, a new cyclic heptapeptide,in rat plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation, has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 μL) in a 96-well plate by methanol (200 μL) containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical column (5 μm, 50 mm × 4.6 mm i.d.) using an eluent of methanol–water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity (r > 0.999) over the quantitation range of 0.1–5 ng/mL. The validation results demonstrated that this method was significantly specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50, and 100 μg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic studies on PK13.     

Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl-l-glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of ¹³C₅ labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018–14 μg FA/g of fresh bread (r ² = 0.997) and 9.3–900 ng 5-MTHF/g of fresh bread (r ² = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79–110 μg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200–300 μg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors.     

Rapid quantification of tilidine,nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r ² > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Quantitative analysis of hair samples for 1-benzylpiperazine (BZP) using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) detection

Benzylpiperazine (BZP) is an amphetamine-type stimulant, which was legally available in New Zealand and widely used in “Party Pills” until reclassification as a Class C drug in April 2008. BZP was included as part of a multi-analyte method developed for hair screening using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS). A 20-mg sample of hair is extracted and partially purified using mixed-mode solid-phase extraction cartridges prior to analysis by LC-MS/MS. The method was developed as a broad screen for drugs of abuse (including amphetamines, opiates, and benzodiazepines), with only the BZP results being presented here. The assay was validated and found to be linear over the range of 0.085 to 8.65 ng/mg with correlation coefficient of r ² ≥ 0.99. Blank hair samples spiked with BZP at 0.22 and 2.16 ng/mg gave intra- and inter-day precision coefficients of variation of ≤10% (n = 6 per day, 3 days) at both levels and calculated extraction efficiencies of 78% and 91%, respectively. The results from the samples submitted to the laboratory for BZP analysis showed 11% were positive (n = 126). The mean BZP level was 3.9 ng/mg (range, 0.4–33 ng/mg; the result was extrapolated when above the calibration). These data are the first available showing the levels expected from users of BZP.     

Development of an LC-MS/MS method for the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder

Serotonin is a major neurotransmitter and affects various functions both in the brain and in the rest of the body. It has been demonstrated that altered serotinergic function is implicated in various psychiatric disorders including depression and schizophrenia. Serotonin has also been implicated along with dopamine in attention deficit–hyperkinetic disorder (AD-HKD). This study provides a versatile validated method for the analysis of serotonin, hydroxyindole acetic acid and dopamine in urine using LC-MS/MS. This method was then used to quantify these analytes in a test group of 17 children diagnosed with severe AD-HKD. This group was compared to a matched control group to investigate the possibility that one of these compounds may be a potential biomarker for this condition. The developed method provided good linear calibration curves for the multiplex assay of analytes in urine (0.05–3.27 nmol/L; R ² ≥ 0.9977). Acceptable inter-day repeatability was achieved for all analytes with RSD values (n = 9) ranging from 1.1% to 9.3% over a concentration range of 0.11–3.27 μmol/L in urine. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 8.8–18.2 nmol/L and the LOQs of 29.4–55.7 nmol/L for analytes in urine. Recoveries were in the ranges of 98–104%, 100–106% and 91–107% for serotonin, 5-HIAA and dopamine, respectively. An appropriate sample clean-up procedure for urine was developed to ensure efficient recovery and reproducibility on analysis. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. Confirmatory analysis was carried out on a linear trap quadrupole-Orbitrap mass spectrometer to obtain high mass accuracy data of the target analytes in the clinical samples.     

Simultaneous quantification of loureirin A and loureirin B in rat urine, feces, and bile by HPLC-MS/MS method and its application to excretion study

A simple HPLC-MS/MS method for simultaneous determination of loureirin A and loureirin B in rat urine, feces, and bile after oral administration of 10.6 g/kg of longxuejie (one rare traditional Chinese medicinal herb) was developed for the first time. The analytes and buspirone (internal standard) were separated on a C₅ column with acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.4 min/mL. The detector was a Q-trap™ mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. Calibration curves of loureirin A in rat urine, feces, and bile were linear over the concentration range of 1.00–5,000 ng/mL. Loureirin B in rat urine, feces, and bile ranged between 0.08 and 20, 0.20 and 20, and 0.10 and 500 ng/mL, respectively. Validation revealed that the method was specific, accurate, and precise. The fully validated method was applied to the excretion study of loureirin A and loureirin B in rats. After oral administration of 10.6 g/kg longxuejie, cumulative excretion amount of loureirin A and loureirin B in rat urine were 2.94 ± 0.81 and 0.36 ± 0.16 μg at 72 h, respectively. Of the total dose, 5.35% of loureirin A and 5.46% of loureirin B were excreted from feces at 60 h. The cumulative amounts of loureirin A and loureirin B in rat bile reached 4.49 ± 0.98 and 5.11 ± 0.83 μg, respectively, at 36 h after dosing, accounting for 0.054% and 0.056% of the total dose.     

Simultaneous online SPE-LC-MS/MS quantification of six widely used synthetic progestins in human plasma

Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography–tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5–100 ng/ml for dienogest, 1–100 ng/ml for etonogestrel and 2–100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from −3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community.     

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d₆ as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d₆ were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d₆. The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d₆ was to just compensate the variation of the injections. The detection limit was 5 ng g⁻¹ swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.     

Semi-automated liquid chromatography-mass spectrometry (LC-MS/MS) method for basic pesticides in wastewater effluents

Effluent from wastewater treatment plants have been identified as an important source of micro-organic contaminants in the environment. An online high-performance liquid chromatography–heated electrospray ionization tandem mass spectrometric method was developed and validated for the determination of basic pesticides in effluent wastewaters. Most available methods for pesticide analysis of wastewater samples are time-consuming, require complex clean-up steps and are difficult to automate. The method developed used a simple solid-phase extraction clean-up for salt and lipid reduction. On-line sample pre-concentration was performed using a reversed phase (C₁₈) column, and analytes were separated by back-flushing onto an analytical column (C₈) with detection using QqQ MS. An option to increase MS resolution was exploited to minimize interference from endogenous compounds in the matrix. A better than unit mass resolution was used (Q1 full width half maximum (FWHM) = 0.2 Da and Q3 FWHM = 0.7 Da), which was as rugged as a unit resolution method, and improved signal/noise and better detection limits were achieved for the targeted basic pesticides. This method was applied to the determination of 11 pesticides, including methoxytriazine, chlorotriazines, chloroacetanilides, phenylurea and carbamate pesticides. The percentage recovery values for these pesticides using the online trapping column were within the range, 73–95%, with relative standard deviation (RSD) values <8.9%. The highest concentrations of these pesticides in wastewater effluents in County Cork, Ireland, were simazine (0.51 μg/L), prometon (0.14 μg/L), diuron (0.21 μg/L) and atrazine (0.19 μg/L).     

GC/MS detection of central nervous tissues as specified BSE risk material in meat products: validation by an externally controlled blind trial

The addition of central nervous tissues (CNT), such as brain and spinal cord, in the manufacturing of meat products is either forbidden—if the material falls under the legal definition of specified risk material (SRM)—or must be labelled on the packed product. To foster official food control, several CNT detection methods were developed, but only fatty acid patterns as detected by gas chromatography/mass spectrometry (GC/MS) allow the further characterization of the detected CNT as to both the animal species and—surprisingly—the age of the animal from which the CNT was derived in accordance with the legal definition. Complementing a previous report in this journal by Lücker et al. 2010 (doi:) on CNT quantification by GC/MS, we now report results of the validation of this new analytical approach by an externally controlled blind trial elucidating its potential to identify the species and age of the CNT detected. The 72 samples (24 standards of emulsion-type sausage, each heated in three different batches: 75°C, 30 min; 115°C, 25 min; 133°C, 40 min) containing porcine, ovine or bovine muscle tissue and differing amounts of CNT (bovine or ovine brain: 0, 0.1, 0.5, 1.0, 3.0% m/m) were produced externally and provided blind for analyses to our laboratory. In accordance with the previous study, heating had no detectable effect on the GC/MS analysis. Judged by the present sensitivity of this method (cut-off 0.2% CNT), all of the samples containing 0.5% or more CNT (n = 57, 100%) were identified correctly as CNT-positive. The CNT species was identified correctly in 54 samples (94.7%), with three samples of one specific standard (0.5% ovine CNT) falsely classified as bovine CNT. However, the CNT age of these samples was correctly classified (more than 12 months). Overall, 57 samples (100%) were correctly classified as SRM-positive and 15 samples (100%) as SRM-negative. To the best of our knowledge, this is the first time that a legal demand for the (1) detection of traces of a specific tissue in a food matrix, (2) the identification of its taxonomic origin and (3) the classification of its age has been shown to be analytically possible in totally blind samples. The very positive validation results of this externally controlled blind trial recommend the present GC/MS approach for the detection of CNT in meat products as a reference method. However, our results also demonstrate the need for further studies, in particular to increase sensitivity and to conduct ring trials including more than one laboratory.     

Determination of low-level ink photoinitiator residues in packaged milk by solid-phase extraction and LC-ESI/MS/MS using triple-quadrupole mass analyzer

A confirmatory and quantitative method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) has been developed for simultaneous determination of seven photoinitiator residues: benzophenone, (1-hydroxycyclohexyl)phenylketone (Irgacure 184), isopropylthioxanthone (ITX), 2-ethylhexyl-(4-dimethylamino)benzoate (EHA or EHDAB), 2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone (Irgacure 907), (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TPO) and 2-benzyl-2-(dimethylamino)-1-(4-morpholinophenyl)-1-butanone (Irgacure 369) in packaged milk and related packaging materials. Residues of photoinitiators were extracted from milk using acetonitrile, and further enriched and purified on HLB solid-phase extraction cartridges prior to being analyzed by LC-ESI/MS/MS with selected reaction monitoring mode, while photoinitiators in packaging materials were extracted using the same solvent. Satisfactory recovery (from 80 to 111%), intra- and inter-day precision (below 12%), and low limits of quantification (from 0.1 to 5.0 μg kg⁻¹) were evaluated from spiked samples at three concentration levels (5.0, 10.0 and 25.0 μg kg⁻¹ for Irgacure 184 and 2.5, 5.0 and 25.0 μg kg⁻¹ for others). These excellent validation data suggested the possibility of using the LC-ESI/MS/MS method for simultaneous determination of low-level photoinitiator residues migrating from printed food-packaging materials into milk. The method has been successfully applied to the analysis of real samples of different fat contents ranging from 8 to 30 g L⁻¹. The photoinitiator residues were revealed to be higher in milk with higher fat content and the most important contaminations were benzophenone and ITX in concentration ranges of 2.84–18.35 and 0.83–8.87 μg kg⁻¹, respectively.     

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.     

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.     

Automated on-line in-tube solid-phase microextraction coupled with HPLC/MS/MS for the determination of butyrophenone derivatives in human plasma

This paper describes a fully automated on-line method combining in-tube solid-phase microextraction (SPME) in which sample clean-up and enrichment are conducted through an open tubular fused-silica capillary column and high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) detection for the determination of six butyrophenone derivatives (moperone, floropipamide, haloperidol, spiroperidol, bromperidol, and pimozide) in human plasma samples. The six butyrophenones were extracted by repeatedly aspirating and dispensing plasma sample solutions on a DB-17 capillary column (60 cm × 0.32 mm i.d., film thickness 0.25 μm). The analytes retained on the inner surface of the capillary column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. Extraction efficiencies ranged from 12.7% to 31.8% for moperone, spiroperidol, and pimozide, and from 1.08% to 4.86% for floropipamide, haloperidol, and bromperidol. The regression equations for all compounds showed excellent linearity, ranging from 0.05 to 50 ng/0.1 mL of plasma, except for moperone and spiroperidol (0.01 to 50 ng/0.1 mL). The limits of detection and quantification in plasma for each drug were 0.03–0.2 and 0.1–0.5 ng/mL, respectively. The intra- and inter-day coefficients of variation for all compounds in plasma were not greater than 13.7%.     

A sensitive LC-MS/MS method for quantification of phenytoin and its major metabolite with application to in vivo investigations of intravenous and intranasal phenytoin delivery

Phenytoin is a powerful antiseizure drug with complex pharmacokinetic properties, making it an interesting model drug to use in preclinical in vivo investigations, especially with regards to formulations aiming to improve drug delivery to the brain. Moreover, it has a major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, which can be simultaneously studied to achieve a better assessment of its behaviour in the body. Here, we describe the development and validation of a sensitive LCMS/MS method for quantification of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain which can be used in such preclinical studies. Calibration curves produced covered a range of 7.81 to 250 ng/mL (plasma) and 23.4 to 750 ng/g (brain tissue) for both analytes. The method was validated for specificity, sensitivity, accuracy, and precision and found to be within the acceptable limits of ±15% over this range in both tissue types. The method when applied in two in vivo investigations: validation of a seizure model and to study the behaviour of a solution of intranasally administered phenytoin as a foundation for future studies into direct nose-to-brain delivery of phenytoin using specifically developed particulate systems, was highly sensitive for detecting phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain.     

Automated mass spectrometric analysis of urinary and plasma serotonin

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R² > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.