Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.     

Determination of thromboxanes,leukotrienes and lipoxins using high-temperature capillary liquid chromatography–tandem mass spectrometry and on-line sample preparation

An on-line strong cation-exchange (SCX)–reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B₂, TXB₃, leukotriene (LT) B₄, LTD₄ and lipoxin (LX) A₄ in cell culture supernatants was developed and validated. In the present method, a high temperature (70 °C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Limits of detection in the range of 1–4 ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB₂ was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.     

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.     

Derivatization procedures for detection of prostaglandins in biological matrices by liquid chromatography/electrochemistry

Conventional reversed-phase HPLC conditions have been optimized for resolution of a mixture containing prostaglandins PGE(1), PGE(2), PGF(1alpha), and PGF(2alpha). Electroactive derivative-forming reagents, such as p-nitrobenzyloxyamine, 2-bromo-2'-nitroacetophenone, and 2,4-dinitrophenylhydrazine have been evaluated for use as precolumn reagents for forming prostaglandin derivatives. The results indicate that detection limits of 120 pg are achievable with amperometric detection. The utility of the procedures developed is illustrated by the detection of prostaglandins in human urine and plasma.     

Simultaneous analysis of prostanoids using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.     

Gas chromatographic-mass spectrometric profiling with negative-ion chemical ionization detection of prostaglandins and their 15-keto and 15-keto-13,14-dihydro catabolites in rat blood

A method was set up in which the primary prostaglandins (PGF2 alpha, PGD2, PGE2, thromboxane B2, 6-keto-PGF1 alpha and 6-keto-PGE1) and their catabolites (15-keto and 15-keto-13,14-dihydro) could be analyzed in the same sample at the same time. The method makes use of long capillary columns (60 m) to resolve the complex mixture during gas chromatography and mass fragmentography to provide the specificity of detection of these products. Selectivity and sensitivity is provided through use of appropriate derivatives (pentafluorobenzyl esters) which allow detection by negative-ion chemical ionization in which high-abundance fragments in the high end of the mass spectrum (M-pentafluorobenzyl) are observed. A purification procedure of whole blood is described involving diethyl ether extraction, C18 Sep-Pak chromatography, derivatization into the pentafluorobenzyl-O-methyloxime, C18 Sep-Pak and silicic acid chromatography followed by final derivatization into trimethylsilyl ethers for gas chromatographic-mass spectrometric analysis. Recovery of added [3H]PGF2 alpha was 73.8 +/- 2.2% (n = 10). Sample workup and analysis takes ten days for six samples. The method is sufficiently sensitive for the profiling of a 10-ml sample of whole blood (limit approximately 1 pg/ml; 1-pg injection on column).     

Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

Analysis of nuclear proteome in C57 mouse liver tissue by a nano-flow 2-D-LC-ESI-MS/MS approach

The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX)-RPLC-ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX-RPLC-ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy.     

Liquid chromatography-tandem mass spectrometric method for determination of the anti-inflammatory compound vicenin-2 in the leaves of L. ericoides Mart

This paper reports a rapid and sensitive method for determination of the anti-inflammatory compound vicenin-2 in L. ericoides Mart. using liquid chromatography-tandem mass spectrometry. Separation of the compound of interest was performed on a VP-ODS(18) (150 x 2 mm, Shimadzu, Japan) column and a pre-column packed with GPV-ODS C(18) (5 x 2 mm, Sigma-Aldrich, USA) with acetonitrile-water (15:85) mobile phase containing 2% acetic acid using isocratic flow at 0.5 mL/min for 2 min. Multiple-reaction monitoring of vicenin-2 was performed using electrospray positive ionization. The linear calibration curves were generated using a concentration range of 5-2500 ng/mL with correlation coefficients >0.99. The values of limit of detection and limit of quantitation were found to be 1 and 5 ng/mL, respectively. The method developed based on LC-ESIMS/MS is advantageous because it permits the rapid and selective detection of vicenin-2. Furthermore, the method can be easily applied to the routine analysis of vicenin-2 in plant extracts using a minimal amount of sample.     

液相色谱-质谱/质谱法对多种食品基体中三聚氰胺的检测

采用超声、振荡、液液萃取、离心等方法提取14种复杂食品基体中的三聚氰胺,提取液经阳离子交换固相萃取柱净化后,采用液相色谱-质谱/质谱法测定多种食品基体中的三聚氰胺.涉及的食品基体包括豆类制品、饮料、糕点、含乳饼干、鲜蛋、蛋制品和调味品6类基体14种食品.方法的检出限为0.005 ～0.012 5 mg/kg,回收率为75% ～115%,RSD小于18%;定量下限为0.025 ～0.062 5 mg/kg,回收率为84% ～106%,RSD小于10%.中、高浓度添加回收率为82% ～110%,RSD小于12%.方法灵敏、准确、有效.     

Speciation of metal-EDTA complexes by flow injection analysis with electrospray ionization mass spectrometry and ion chromatography with inductively coupled plasma mass spectrometry

Flow injection analysis (FIA) with ESI-MS and ion chromatography (IC) with inductively coupled plasma-MS (ICP-MS) as the complementary technique have been explored for the determination of metal ions as their metal-EDTA complexes. ESI-MS enabled the identification of metal-EDTA complexes such as [Mn(EDTA)](2-), [Co(EDTA)](2-), [Ni(EDTA)](2-), [Cu(EDTA)](2-), [Zn(EDTA)](2-), [Pb(EDTA)](2-), and [Fe(EDTA)](1-) and their MS spectral showed that these metal-EDTA complexes were present in solution. Based on the ESI-MS, ion chromatographic separation and ICP-MS detection of these complexes are possible because IC-ICP-MS requires stable metal-EDTA complex during the chromatographic separation. The separation of these metal-EDTA complexes was achieved on an anion-exchange column with a mobile phase containing 30 mM NH(4)(HPO(4))(2) at pH 7.5 within 7 min with ICP-MS providing element specific detection. The ICP-MS LODs for the metal-EDTA were in the range of 0.1-0.5 microg/L with the exception of Fe (15 microg/L). The proposed method was a simple procedure for sample processing, using direct injection of sample without removal of sample matrix and was successfully applied to the determination of metal-EDTA complexes in real samples.     

Identification of reaction products from the pyridinium dichromate derivatization of prostaglandins by high-performance liquid chromatography and direct chemical ionization mass spectrometry

The reaction products from the oxidation of prostaglandins with pyridinium dichromate have been identified by direct chemical ionization mass spectrometry of the underivatized compounds after separation by reversed-phase high-performance liquid chromatography and ultraviolet diode-array detection. The thermal influence on the reproducibility of the dehydration patterns of the mass spectra was studied. The main products from the prostaglandins E1, E2, F1 alpha and F2 alpha were the corresponding 15-oxo derivatives. Minor amounts of the 9,11,15-trioxoprostaglandin were formed from PGE, while the oxidation of PGF was less selective, yielding additional dioxo derivatives. Addition of water to the reagent reduced the reactivity, but increased the selectivity in favour of the formation of 15-oxo-PGF during the oxidation of PGF.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

固相萃取/液相色谱-串联质谱法测定水中苯胺类化合物及基质干扰的消除

建立了固相萃取/液相色谱-串联质谱检测水中18种苯胺类化合物的分析方法,并优化了固相萃取和色谱条件。水样经混合型阳离子交换柱(MCX)或硅胶基体阳离子交换柱(SCX)富集后,用氨水甲醇溶液洗脱,用超纯水适当地稀释后,用液相色谱-串联质谱法测定。以ODS柱为分离柱,甲醇-0.005%(v/v)甲酸水溶液为流动相,梯度洗脱,多反应监测模式分析,内标法定量。18种苯胺类化合物的分析时间在15 min之内。采用MCX柱萃取时,16种苯胺化合物的方法检出限为0.002~0.035 μ g/L,地表水样品的加标回收率为72.5%~92.5%,相对标准偏差为1.4%~9.6%;采用SCX柱萃取时,17种苯胺类化合物的方法检出限为0.013~0.207 μ g/L,地表水样品的加标回收率为66.5%~102%,相对标准偏差为2.4%~13.6%。本实验还考察了消除基质干扰的5种方法,结果表明,调整色谱分离条件是最有效的方法,其次是选择合适的前处理方法。更换离子源、内标法定量和利用基质标准溶液校正也可在一定程度上消除或补偿基质干扰。     

Isoform-specific quantification of endothelins in HUVEC culture supernatants by on-line high-performance liquid chromatography/electrospray mass spectrometry

A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific and employs solid-phase extraction and subsequent HPLC fractionation followed by HPLC-ESIMS analysis. The peptide vasoactive-intestinal-contractor (VIC) was used as internal standard for the HPLC-ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC-ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin-1 (ET1) were observed. Basal levels were determined to be 1.8 +/- 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method.     

Determination of lovastatin in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A fast and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of lovastatin in human plasma. With simvastatin as internal standard, sample pretreatment involved one-step extraction with n-hexane-methylene dichloride-isopropanol (20:10:1, v/v/v) of 0.5 mL plasma. Chromatographic separation was carried out on an Acquity UPLC BEH C(18) column with mobile phase consisting of acetonitrile-water (containing 5 mmol/L ammonium acetate; 85:15, v/v) at a flow-rate of 0.35 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization source with positive mode. The analysis time was shorter than 1.7 min per sample. The standard curve was linear (r2>or=0.99) over the concentration range 0.025-50.0 ng/mL with a lower limit of quantification of 0.025 ng/mL. The intra- and inter-day precision values were below 11% and the accuracy (relative error) was within 6.0% at three quality control levels. This is the first method of MS with MRM coupled to UPLC for the determination of lovastatin, which showed great advantages of high sensitivity, selectivity and high sample throughput. It was fully validated and successfully applied to the pharmacokinetic study of lovastatin tablets in healthy Chinese male volunteers after oral administration.     

Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.     

Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation‐exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)–tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive‐mode APCI. Responses for all analytes were linear over the calibration ranges. Intra‐assay precision (RSD) was 2–18%, and inter‐assay precision was 2–12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC₅₀) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC₅₀ of 120 μg L⁻¹ for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L⁻¹ in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.     

Measurement of quetiapine and four quetiapine metabolites in human plasma by LC-MS/MS

There is interest in monitoring plasma concentrations of N‐desalkylquetiapine in relation to antidepressant effect. A simple LC‐MS/MS method for quetiapine and four metabolites in human plasma (50 μL) has been developed to measure concentrations of these compounds attained during therapy. Analytes and internal standard (quetiapine‐d8) were extracted into butyl acetate–butanol (10:1, v/v) and a portion of the extract analysed by LC‐MS/MS (100 × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow‐rate 0.5 mL/min; positive ion APCI‐SRM, two transitions per analyte). Assay calibration (human plasma calibrators) was linear across the ranges studied (quetiapine and N‐desalkylquetiapine 5–800, quetiapine sulfoxide 100–15,000, others 2–100 µg/L). Assay validation was as per FDA guidelines. Quetiapine sulfone was found to be unstable and to degrade to quetiapine sulfoxide. In 47 plasma samples from patients prescribed quetiapine (prescribed dose 200–950 mg/day), the (median, range) concentrations found (µg/L) were: quetiapine 83 (7–748), N‐desalkylquetiapine, 127 (7–329), O‐desalkylquetiapine 12 (2–37), 7‐hydroxyquetiapine 3 (<1–48), and quetiapine sulfoxide 3,379 (343–21,704). The analyte concentrations found were comparable to those reported by others except that the concentrations of the sulfoxide were markedly higher. The reason for this discrepancy in unclear. Copyright © 2012 John Wiley & Sons, Ltd.