Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.     

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.     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.     

Rapid and Sensitive LC–MS/MS Analysis of Fatty Acids in Clinical Samples

Free fatty acids (FFAs), major cellular metabolites, play an important role during tumor pathogenesis. Enhanced de novo fatty acid synthesis in tissues is a characteristic feature of cancer. Therefore, measurement of FFA concentration in biological samples is beneficial for cancer research and clinical diagnosis. Herein, a rapid, stable, and sensitive detection methodology was established to simultaneously quantify 22 FFAs using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS). The HPLC–MS/MS system was run in negative ion mode for 15 min using multiple reaction monitoring. The lipids were extracted from colon tissues of colon cancer patients and then injected into the HPLC–MS/MS system for analysis. Colon samples were analyzed by inter-day repeatability and intra-day repeatability, with less than 5 % deviation for most fatty acids. This approach is successful to determine low picogram concentrations of each FFA molecule using milligrams of tissue, and provides a promising method for FFA microanalysis in clinical samples.

     

Analysis of aliphatic and aromatic carbonyl compounds in ambient air by LC/MS/MS.

A sensitive liquid chromatograph/tandem mass spectrometric technique (LC/MS/MS) was applied to determine aliphatic and aromatic carbonyl compounds in ambient air. Traces of the carbonyl compounds were sampled by passing through a Sep-Pak DNPH-silica cartridge. Their derivatives were thus eluted with acetonitrile, separated by reversed-phase liquid chromatography and determined by quadrupole tandem mass spectrometry in an atmospheric pressure chemical ionization (APCI) mode with multiple reaction monitoring (MRM). The detection limits (DL) of the carbonyl compounds were 0.8 - 15 ng/m3. A number of the carbonyl compounds were detected at n.d.- 14 microg/m3 levels. The precursor ion scanning analysis was applied to identify the unknown compounds.     

An LC/ESI–MS/MS method to quantify the PI3K inhibitor GDC-0084 in human plasma and cerebrospinal fluid: Validation and clinical application

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) method was developed and validated to measure GDC-0084 in human plasma and cerebrospinal fluid (CSF). Reverse-phase chromatography with gradient elution was performed using a C₁₈ column (50 × 2.0 mm, 3 μm). Solid-phase extraction of plasma and CSF was employed to give excellent recovery. MS detection was performed with positive ion screening in multiple reaction monitoring mode. The precursor to the product ions (Q1 → Q3) selected for GDC-0084 and GDC-0084-d₆ were 383.2 → 353.2 and 389.2 → 353.2, respectively. A separate calibration curve was established for human plasma and CSF. Both calibration curves, ranging from 0.2 to 200 ng/mL, were linear and had acceptable intra- and inter-day precision and accuracy. The lower limit of quantitation and limit of detection for GDC-0084 in human plasma were 0.2 ng/mL (signal/noise ≥47) and 0.005 ng/mL (signal/noise ≥3.5), respectively, and for GDC-0084 in human CSF were 0.2 ng/mL (signal/noise ≥19.7) and 0.04 ng/mL (signal/noise ≥7.2). This method was successfully applied to analyze serial plasma samples obtained from children with diffuse intrinsic pontine gliomas and other midline gliomas who participated in pharmacokinetic studies as part of a phase I clinical trial of GDC-0084.     

超高效液相色谱-串联质谱法测定食品包装材料中全氟辛烷磺酸盐

建立了超高效液相色谱-串联质谱(UPLC- MS/MS)测定食品包装材料中全氟辛烷磺酸盐(PFOS)的方法.采用乙腈作为溶剂,加速溶剂提取法提取食品包装材料中的PFOS.色谱条件:ACQUITY UPLC BEH C₁₈色谱柱（1.7 μm,2.1 mm×50 mm）;柱温:30 ℃;流动相:乙腈/水,梯度洗脱;流速:0.2 mL/min;经UPLC分离后用多级反应监测(MRM)方式测定.用2个子离子的相对丰度定性, 外标法定量.PFOS在0.005～0.500 μg/mL范围内线性良好(R2=0.999),PFOS的回收率为90.0%～101.6%,相对标准偏差RSD为1.5%～3.5%.方法检出限为0.1 μg/m2(S/N≥3).     

ESI outcompetes other ion sources in LC/MS trace analysis

Choosing an appropriate ion source is a crucial step in liquid chromatography mass spectrometry (LC/MS) method development. In this paper, we compare four ion sources for LC/MS analysis of 40 pesticides in tomato and garlic matrices. We compare electrospray ionisation (ESI) source, thermally focused/heated electrospray (HESI), atmospheric pressure photoionisation (APPI) source with and without dopant, and multimode source in ESI mode, atmospheric pressure chemical ionisation (APCI) mode, and combined mode using both ESI and APCI, i.e. altogether seven different ionisation modes. The lowest limits of detection (LoDs) were obtained by ESI and HESI. Widest linear ranges were observed with the conventional ESI source without heated nebuliser gas. In comparison to HESI, ESI source was significantly less affected by matrix effect. APPI ranked second (after ESI) by not being influenced by matrix effect; therefore, it would be a good alternative to ESI if low LoDs are not required.

Graphical abstract

     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Sensitive analysis of metal cations in positive ion mode electrospray ionization mass spectrometry using commercial chelating agents and cationic ion‐pairing reagents

Metals play a very important role in many scientific and environmental fields, and thus their detection and analysis is of great necessity. A simple and very sensitive method has been developed herein for the detection of metals in positive ion mode ESI‐MS. Metal ions are positively charged, and as such they can potentially be detected in positive ion mode ESI‐MS; however, their small mass‐to‐charge (m/z) ratio makes them fall in the low‐mass region of the mass spectrum, which has the largest background noise. Therefore, their detection can become extremely difficult. A better and well‐known way to detect metals by ESI‐MS is by chelating them with complexation agents. In this study eleven different metals, Fe(II), Fe(III), Mg(II), Cu(II), Ru(III), Co(II), Ca(II), Ni(II), Mn(II), Sn(II), and Ag(I), were paired with two commercially available chelating agents: ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS). Since negative ion mode ESI‐MS has many disadvantages compared to positive ion mode ESI‐MS, it would be very beneficial if these negatively charged complex ions could be detected in the positive mode. Such a method is described in this paper and it is shown to achieve much lower sensitivities. Each of the negatively charged metal complexes is paired with six cationic ion‐pairing reagents. The new positively charged ternary complexes are then analyzed by ESI‐MS in the positive single ion monitoring (SIM) and single reaction monitoring (SRM) modes. The results clearly revealed that the presence of the cationic reagents significantly improved the sensitivity for these analytes, often by several orders of magnitude. This novel method developed herein for the detection of metals improved the limits of detection (LODs) significantly when compared to negative ion mode ESI‐MS and shows great potential in future trace studies of these and many other species. Copyright © 2012 John Wiley & Sons, Ltd.     

Screening and analysis of the multiple absorbed bioactive components and metabolites in rat plasma after oral administration of Jitai tablets by high‐performance liquid chromatography/diode‐array detection coupled with electrospray ionization tandem mass spectrometry

Based on the serum pharmacochemistry technique and high‐performance liquid chromatography/diode‐array detection (HPLC/DAD) coupled with electrospray tandem mass spectrometry (HPLC/ESI‐MS/MS), a method for screening and analysis of the multiple absorbed bioactive components and metabolites of Jitai tablets (JTT) in orally dosed rat plasma was developed. Plasma was treated by methanol precipitation prior to liquid chromatography, and the separation was carried out on a Symmetry C₁₈ column, with a linear gradient (0.1% formic acid/water/acetonitrile). Mass spectra were acquired in negative and positive ion modes, respectively. As a result, 26 bioactive components originated from JTT and 5 metabolites were tentatively identified in orally dosed rat plasma by comparing their retention times and MS spectra with those of authentic standards and literature data. It is concluded that an effective and reliable analytical method was set up for screening the bioactive components of Chinese herbal medicine, which provided a meaningful basis for further pharmacology and active mechanism research of JTT. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a precise quantitative method for monitoring sirolimus in whole blood using LC/ESI–MS/MS

Sirolimus is used on patients after solid organ transplantation and on lymphangioleiomyomatosis (LAM) patients, and therapeutic drug monitoring is required in clinical practice. We have previously reported an accurate method for quantitative determination of sirolimus, but its sample preparation step was complicated. In this study, we developed a modified liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) method for sirolimus quantification. A supported liquid extraction cartridge was used to purify sirolimus from whole blood and ion suppression was mostly prevented. The validation results met the acceptance criteria. This method was compared with the antigen conjugated magnetic immunoassay (ACMIA) and our previously reported method, using whole blood samples from LAM patients. Comparison of the Bland–Altman plots of the currently developed method and the previous method revealed no significant difference between the two methods (mean bias, −2.02%; 95% CI, −7.81–3.78). The values obtained using ACMIA were significantly higher than those obtained using the current method by 13.87% (95% CI, 6.49–21.25) owing to cross-reactivity. The degrees of cross reactivities in LAM patients and in organ transplant patients were similar, and our LC/ESI–MS/MS method precisely measured the blood concentrations of sirolimus.     

Analysis of polar hydrophilic aromatic sulfonates in waste water treatment plants by CE/MS and LC/MS

The present work describes the development and optimization of a capillary (zone) electrophoresis/mass spectrometric (CE/MS) analysis method for polar hydrophilic aromatic sulfonates (ASs). The compounds were detected by negative ion electrospray ionization (NIESI) and selected ion monitoring (SIM). In comparison with CE/UV, for CE/MS a lower-concentration volatile ammonium acetate buffer (5 mM) without organic modifier and a higher separation voltage were better suited for separation. Sensitivity of CE/MS was slightly better than for CF/UV, with the limit of detection (LOD) ranging between 0.1 and 0.4 mg l(-1). For verification of the CE/MS results, ASs were also analysed by ion-pair liquid chromatography/diode array UV detection coupled in series with electrospray mass spectrometry (IPC/DAD/ESI-MS). Real water samples of different waste water treatment plants (WWTPs) in Catalonia (NE Spain) were extracted by solid-phase extraction (SPE) with LiChrolut EN and analysed with CE/MS and LC/MS. ASs were found in influent and effluent water samples of the WWTPs in the microg l(-1) concentration range. LC/MS offered a higher separation efficiency and sensitivity than CE/MS. Therefore with LC/MS more compounds could be identified in the WWTPs. The persistency of the ASs was distinct: some compounds were well degraded during the water treatment process, while others were quite persistent.     

高效液相色谱-串联质谱法测定食品包装材料中全氟辛烷磺酰基化合物(PFOS)

建立了用高效液相色谱-串联质谱(HPLC/MS/MS)结合快速溶剂萃取测定食品包装材料中全氟辛烷磺酰基化合物(PFOS)的方法。采用乙腈溶剂,快速溶剂提取食品包装材料中的PFOS,提取液经0.2μm有机滤膜过滤后,以V(乙腈)∶V(10 mmol/L乙酸铵溶液)=80∶20为流动相,经HPLC分离后用多级反应监测(MRM)方式测定。用两个子离子的相对丰度定性,外标法定量。PFOS在0.002~0.1μg/mL范围内线性良好(R2=0.998),回收率为93.8%~101%,精密度RSD为1.6%~3.1%,方法检出限为0.4μg/m2(S/N≥10),满足欧盟法规对食品包装材料中PFOS的限量检测要求。方法可用于食品包装材料中PFOS的检测。     

Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples

Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.     

Determination of pinocembrin in human plasma by solid‐phase extraction and LC/MS/MS: application to pharmacokinetic studies

A sensitive, fast and specific method for the quantitation of pinocembrin in human plasma based on high‐performance liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed and validated. Clonazepam was used as the internal standard (IS). After solid‐phase extraction of 500 μL plasma, pinocembrin and the IS were separated on a Luna C₈ column using the mobile phase composed of acetonitrile–0.3 mm ammonium acetate solution (65:35, v/v) at a flow rate of 0.25 mL/min in isocratic mode. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source in negative mode by AB SCIEX Qtrap 5500. The assay was linear from 1 to 400 ng/mL, with within‐ and between‐run accuracy (relative error) from ?1.82 to 0.54%, and within‐ and between‐run precision (CV) below 5.25%. The recovery was above 88% for the analyte at 1, 50 and 300 ng/mL. This analytical method was successful for the determination of pinocembrin in human plasma and applied to a pharmacokinetic study of pinocembrin injection in healthy volunteers after intravenous drip administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultra‐performance hydrophilic interaction liquid chromatography/tandem mass spectrometry for the determination of everolimus in mouse plasma

Ultra‐performance hydrophilic interaction liquid chromatography (UPHILIC) interfaced with the electrospray ionization (ESI) source of a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of everolimus in mouse plasma samples. UPHILIC was performed on a sub‐2 µm bare silica particle packing with the column pressure under traditional high‐performance liquid chromatography (HPLC) to allow fast separation of pharmaceutical compounds within a chromatographic analysis time of 1 min. This UPHILIC technology is comparable with reversed‐phase ultra‐performance liquid chromatography (RPUPLC) in terms of chromatographic efficiency but demands neither expensive ultra‐high‐pressure instrumentation nor new laboratory protocols. With the ESI source, multiple reaction monitoring (MRM) of the ammoniated adduct ions of the analyte was used for tandem mass spectrometric detection. The retention mechanism profiles of the test compounds under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of the test compounds in positive ion mode were investigated. A UPHILIC/MS/MS approach following a protein precipitation procedure was applied for the quantitative determination of everolimus at the low ng/mL region in support of a pharmacodynamic study. The analytical results obtained by the UPHILIC/MS/MS approach were fond to be in good agreement with those obtained by the RPUPLC/MS/MS method in terms of assay sample throughput, sensitivity and accuracy. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.