Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.     

High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood

Eleven phenothiazine derivatives with heavy side chains contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)/electrospray (ES) tandem mass spectrometry (MS). All compounds gave the base peaks due to [M + 1](+) by HPLC/ES single MS. The product ions formed from each quasi-molecular ion by HPLC/ES tandem MS showed the base peaks due to side chains liberated. The mass chromatography of HPLC/ES tandem MS showed much higher sensitivity than that of HPLC/ES single MS for phenothiazines spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and reproducibility were studied for thiethylperazine, clospirazine and flupentixol spiked to human whole blood by means of mass chromatography of HPLC/ES tandem MS. The three compounds showed good linearity in the range of 2-40 ng/mL with a detection limit of about 0.5 ng/mL. Recoveries of the three compounds spiked to whole blood (2 and 8 ng added to 1 mL whole blood) were 43.4-72.5 %; the coefficients of intraday and interday variations were 3.7-9.3 and 12.6-17.9 %, respectively. Thiethylperazine, clospirazine and flupentixol in whole blood could actually be determined with sufficient sensitivity 3 and 6 h after oral administration of 5-10 mg of each compound in a volunteer.     

离子色谱-三重四极杆质谱法同时测定血浆和尿液中百草枯和敌草快

百草枯和敌草快是广泛使用的非选择性触杀型除草剂,中毒后会造成急性肺损伤,病死率高,同时监测血浆和尿液中百草枯和敌草快的浓度,可以为临床早期诊断和预后提供有价值的信息。血浆和尿液中百草枯和敌草快的主要检测方法为液相色谱-质谱法。百草枯和敌草快为强极性水溶性化合物,在反相色谱柱上难以保留,多采用离子对色谱法或亲水色谱法进行分离。采用离子对色谱法时,加入的离子对试剂有离子抑制作用,降低了质谱检测的灵敏度,还给质谱系统增加了额外的污染;亲水色谱法易受基质成分影响,保留时间不稳定。考虑到百草枯和敌草快在水溶液中以双电荷联吡啶离子状态存在,更适合采用阳离子交换色谱法,建立了离子色谱-三重四极杆质谱测定血浆和尿液中百草枯和敌草快的检测方法。血浆和尿液样品经水稀释后,直接过混合型聚合物反相吸附和弱阳离子交换固相萃取柱（Oasis WCX）净化,经IonPac CS 18型阳离子色谱柱（250 mm×2.0 mm,6.0 μm）分离,以自动在线产生的甲磺酸进行梯度洗脱,色谱柱流出液经阳离子抑制器抑制后进入质谱系统,在ESI⁺ 、多反应监测（MRM）模式下检测,稳定同位素内标法定量。百草枯和敌草快分别在1.0~150 μg/L和0.5~75 μg/L范围内线性关系良好,血浆中的平均基质效应分别为84.2%~89.3%和84.7%~91.1%,尿液中平均基质效应分别为50.3%~58.4%和51.9%~59.4%;血浆中百草枯和敌草快的平均加标回收率分别为93.5%~117%和91.7%~112%,相对标准偏差（RSD）分别为3.4%~16.7%和2.8%~13.2%;尿液中百草枯和敌草快的平均加标回收率分别为90.0%~118%和99.2%~116%,RSD分别为5.6%~14.9%和2.4%~17.3%（n =6）;血浆和尿液中百草枯和敌草快的检出限分别0.3 μg/L和0.2 μg/L,定量限分别为1.0 μg/L和0.5 μg/L。该法灵敏度高,准确性好,可用于血浆和尿液中百草枯和敌草快的中毒检测。     

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.     

Monolithic spin column extraction and GC-MS for the simultaneous assay of diquat,paraquat, and fenitrothion in human serum and urine

We present a method based on monolitic spin column extraction and gas chromatography–mass spectrometry as an analytical method for screening diquat (DQ), paraquat (PQ), and fenitrothion in serum and urine. This method is useful for clinical and forensic toxicological analyses. Recovery of DQ, PQ, and fenitrothion from serum and urine, spiked at concentrations between 0.1, 2.5, 20, and 45 μg/ml, ranged from 51.3% to 106.1%. Relative standard deviation percentages were between 3.3% and 14.8%. Detection and quantitation limits for serum and urine were 0.025 and 0.05 μg/ml, respectively, for DQ, 0.1 and 0.1 μg/ml, respectively, for PQ, and 0.025 and 0.05 μg/ml, respectively, for fenitrothion. Therefore, these compounds can be detected and quantified in the case of acute poisoning.     

Feasibility of static secondary ion mass spectrometry to study physicochemical interactions between organic components and silver in thermographic systems

Chemical engineering of high-technology products requires elucidation of intermolecular interactions in complex materials. As part of an extensive study on thermographic systems, static secondary ion mass spectrometry (S-SIMS) was used to probe the physicochemical behaviour of active compounds, such as different tone modifiers and stabilisers, on silver. In particular, the feasibility of detecting adsorption and/or binding of individual additives and mixtures to silver was examined. Substrates prepared by sputter coating silver on silicon wafers were exposed to solutions of the studied compounds in 2-butanone. The signal intensities measured with S-SIMS for the ad-layers showed reproducibility to within 10%. Radical ions containing silver such as [M-H+Ag]+ * were used as evidence for the formation of bonds in the solid. Also the [M-H+2Ag]+ ions could be assigned to chemisorbed species while [M+Ag]+ ions could be formed by adduct ionisation of molecules with co-ejected Ag+ ions. The signal intensities of [M-H+Ag]+ * and [M-H+2Ag]+ ions were used to monitor the adsorption quantitatively as a function of time.     

A non-covalent dimer formed in electrospray ionisation mass spectrometry behaving as a precursor for fragmentations

A non-covalent-bonded dimer was detected in the positive ion electrospray ionisation (ESI) mass spectra of a synthetic impurity. In tandem mass spectrometry (MS/MS) experiments using collision-induced dissociation (CID), the ion was found to behave as a [M+H]+-type precursor ion for fragmentation until MS5. The dimer was probably formed through multi-hydrogen bonds over a proton bridge. When the fragmentation occurred at the center of the bridge, the dimer was broken apart to give monomer fragments at MS6. However, no corresponding deprotonated dimer [2M-H]- was found in the negative ion ESI spectra. The dimer was extremely stable, and it could still be observed when a fragmentation voltage of up to 50 V was applied in the ionisation source. The formation of the non-covalent dimer was also found to be instrument-dependent, but independent of sample concentration. Accurate mass measurements of the [2M+H]+ and [M+H]+ ions, and their MSn product ions, provided the basis for assessing the fragmentation mechanism proposed for [2M+H]+. The fragmentation pathway was also illustrated for the deprotonated molecule [M-H]-.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry

The phenolic components from Radix Salvia miltiorrhizae Bunge, a well-known herbal medicine (Dan-Shen in Chinese), have been investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the ESI mass spectra a predominant [M-H]- ion was observed in negative mode and provided molecular mass information. ESI-MS/MS spectra of the [M-H]- ions were used for structural analysis, based on the spectra of standards. It was found that caffeic acid and its monomeric analogs containing a carboxyl group readily lost CO2, while dimers, trimers and tetramers of caffeic acid expelled successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in S. miltiorrhizae were characterized, of which eight compounds were positively identified by comparison with standards. The remaining twenty phenolics for which standards were not available were tentatively identified based on their UV spectra and MS/MS fragmentation characteristics.     

Separation and quantification of paraquat and diquat in serum and urine by capillary electrophoresis

The use of capillary electrophoresis (CE) for simultaneous qualitative and quantitative detection of paraquat (PQ) and diquat (DQ) in both serum and urine was investigated. The two herbicides were extracted from biological fluids with liquefied phenol. Serum required a deproteinization with chloroform and ammonium sulfate as pretreatment. The extracts were hydrodynamically injected and the complete separation was carried out in 10 min, using a capillary tube (75 microm i.d., 500 mm) of fused silica containing 50 mM phosphate buffer (pH 2.50) as the carrier. UV absorbance detection at 200 nm was performed by an on-column detector. The analytes were characterized by their respective migration times. Analytical recoveries were 52.6% for PQ and 62.6% for DQ in serum, and 71.4% and 59.3%, respectively, in urine. The linearity was studied up to 4 mg/L and the limits of detection (LODs) were better than 5 pg/mL in serum or urine. The CE method described was applied to the characterization of two lethal poisonings and results were related.     

固相萃取净化-超高效液相色谱-高分辨质谱法测定尿液中百草枯和敌草快残留

尿液样品中百草枯(PQ)和敌草快(DQ)的检测是理化检验工作的难点。PQ和DQ具有分子极性大和水溶性好等特点,常规反相色谱柱难以保留;现有文献方法多采用亲水相互作用色谱法(HILIC)进行保留,但文献方法需采用高浓度缓冲盐作为流动相,增加了质谱仪的污染。基于上述问题,研究建立了弱阳离子交换(WCX)固相萃取净化-超高效液相色谱-高分辨质谱法(UPLC-HRMS)快速准确测定尿液样品中PQ和DQ残留的检测方法。尿液样品经混合磷酸盐缓冲液(pH=6.86)稀释和WCX固相萃取净化后,在Syncronis HILIC色谱柱(100 mm×2.1 mm, 1.7 μm)上进行梯度洗脱分离,采用正离子电喷雾离子化模式(ESI⁺)和一级全扫描-数据依赖二级质谱扫描模式(Full mass-ddMS²)进行定量分析。研究通过对色谱条件的不断优化,将HILIC模式下流动相中甲酸铵缓冲盐的浓度降低至10 mmol/L,并系统优化了样品前处理过程中影响PQ和DQ准确性的因素。在最优条件下,PQ和DQ线性关系良好(r²>0.998),在4个加标水平下(1.0、20.0、100.0和200.0 μg/L), PQ和DQ的平均加标回收率分别为85.8%~101%和80.3%~86.9%,精密度(RSD)分别为0.8%~5.1%和0.9%~4.2%。方法的检出限(S/N≥3)和定量限(S/N≥10)分别为0.2 μg/L和0.6 μg/L。将建立的方法用于中毒病人临床治疗过程尿液中DQ含量的跟踪监测。该方法具有快速、简便、灵敏和准确等优点,适用于临床中毒病例尿液样品中PQ和DQ的检测。     

Quantification of paraquat in postmortem samples by gas chromatography-ion trap mass spectrometry and review of the literature

Paraquat (PQ) is an herbicide implicated in numerous fatalities, mainly caused by voluntary ingestion. Several methods have been used to quantify PQ in plasma and urine samples of intoxicated humans as a predictor of clinical outcome. There is no validated method for the analysis of PQ in postmortem samples. Therefore, the aim of this study was to develop an analytical method, using gas chromatography–ion trap mass spectrometry (GC‐IT/MS) after solid‐phase extraction, to quantify PQ in postmortem samples, namely in whole blood, urine, liver, lung and kidney, to cover the routes of distribution, accumulation and elimination of PQ. The method proved to be selective as there were no interferences of endogenous compounds with the same retention time as PQ and ethyl paraquat (internal standard). The regression analysis for PQ was linear in the range 0–10 µg/mL. The detection limits ranged from 0.0076 µg/mL for urine to 0.047 µg/mL for whole blood, and the recoveries were suitable for forensic analysis. The proposed GC‐IT/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of PQ in postmortem samples. The proof of applicability was performed in two fatal PQ intoxications. A review of the analytical methods for the determination of quaternary ammonium herbicides is also provided for a better understanding of the presently available techniques. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid determination of three anticoagulant rodenticides in whole blood by liquid chromatography coupled with electrospray ionization mass spectrometry

A rapid, sensitive and selective method for the simultaneous determination of bromadiolone, flocoumafen and brodifacoum in whole blood using warfarin as internal standard (IS) by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) has been developed and validated. The target compounds were extracted from the whole blood with ethyl acetate and separated on an XDB C18 column (150 mm x 2.1 mm i.d. x 5 microm) by using a mobile phase consisting of 0.2% acetic acid/methanol (12/88, v/v) at a constant flow rate of 0.50 mL/min. The analytes were detected using negative ESI-MS in the selected ion monitoring (SIM) mode. The molecular ions [M-H]- of m/z 527, 541,523 and 307 were selected for the quantification for bromadiolone, flocoumafen, brodifacoum and the IS, respectively. The calibration curves were linear (r2 > 0.995) in the concentration range of 0.50-100.00 ng/mL. The method showed a satisfactory sensitivity (0.05-0.5 ng/mL using 200 microL blood), precision (RSD < 11.9%), accuracy (recovery: 82.0-96.1%) and selectivity. This method was successfully applied to the determination of the analytes for the diagnoses of poisoned human beings and animals.     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Sensitive determination of n-hexane and cyclohexane in human body fluids by capillary gas chromatography with cryogenic oven trapping

A sensitive method was developed for determination of n-hexane and cyclohexane in human body fluids by headspace capillary gas chromatography (GC) with cryogenic oven trapping. Whole blood and urine samples containing n-hexane and cyclohexane were heated in a 7.5 mL vial at 70 degrees C for 15 min, and 5 mL of the headspace vapor was drawn into a glass syringe. All vapor was introduced through an injection port of a GC instrument in the splitless mode into an Rtx-Volatiles middle-bore capillary column at an oven temperature of -40 degrees C for trapping volatile compounds. The oven temperature was programmed to 180 degrees C for GC with flame ionization detection. These conditions gave sharp peaks for both n-hexane and cyclohexane, a good separation of each peak, and low background impurities for whole blood and urine. The extraction efficiencies of n-hexane and cyclohexane were 13.2-30.3% for whole blood and 12.7-20.7% for urine. The coefficients of within-day variation in terms of extraction efficiency of both compounds were 5.0-9.5% for whole blood and 3.8-10.8% for urine; those of day-to-day variation for the compounds were not greater than 16.6%. The regression equations for n-hexane and cyclohexane showed good linearity in the range of 5-500 ng/0.5 mL for whole blood and urine. The detection limits (signal-to-noise ratio = 3) for both compounds were 1.2 and 0.5 ng/0.5 mL for whole blood and urine, respectively. The data on n-hexane or cyclohexane in rat blood after inhalation of each compound are also presented.     

Sensitive analysis of aconitine, hypaconitine, mesaconitine and jesaconitine in human body fluids and Aconitum tubers by LC/ESI-TOF-MS

The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H]+ ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology.     

Simultaneous determination of the herbicides diquat and paraquat in water

A method based on solid-phase extraction (on silica cartridges) and high-performance liquid chromatography (HPLC) followed by diode array UV detection is presented as an analytical tool for screening diquat (DQ) and paraquat (PQ) in drinking waters. The method is useful for quality control laboratories of water companies and beverage industries. Absolute recoveries of DQ and PQ from drinking water (25 mL in all cases), spiked at levels between 0.1, 1.0, and 5.0 microg/L, range from 91% to 103%. Relative standard deviation percentages are between 3% and 11%. Quantitation and detection limits are 70 and 40 ng/L for DQ and 90 and 60 ng/L for PQ, respectively; therefore, these herbicides can be detected and quantitated at levels below the limits established by the European Union.     

Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M H]^ ,[M Na]^ and [M-H]^- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.     

液相色谱-大气压化学电离质谱法分析人参中的人参皂甙

研究了用反相高效液相色谱-大气压化学电离质谱(HPLC/APCI-MS)分析人参皂甙的方法。液相色谱采用乙腈-水流动相进行梯度洗脱,质谱采用正负离子同时扫描并结合二级质谱进行定性,用选择反应离子模式(SRM)测定检测限。实验发现虽然人参皂甙是热不稳定物质,但在大气压化学电离质谱的高温汽化过程中仍能检测到很强的负离子分子离子峰,而且随着汽化温度的升高,人参皂甙的负离子分子离子峰的强度增加。该方法对人参皂甙Rb1和Rg1的检测限分别为1.2×10-13 g和3.0×10-14 g,并检测出白参中包括丙二酰人参皂甙在内的29种人参皂甙。该法灵敏度高,重复性好,结果准确,能有效地对药材提取物中的多种人参皂甙进行检测和结构分析。