Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.     

Application of LC‐MS and LC‐MS‐MS to the analysis of photo‐decomposed crystal violet in the investigation of cultural heritage materials aging

In this work, the accurate liquid chromatography‐ultraviolet‐visible (LC‐UV‐Vis), LC‐mass spectrometry (MS) and LC‐MS‐MS analysis of the photo‐degradation products of crystal violet (CV) is reported. CV is a light fugitive early synthetic dye which had a widespread diffusion into the market starting from the end of the XIX century and was used among others by V. Van Gogh and P. Gauguin in their writings, drawings or paintings. On‐line photodiode array detector enabled simultaneous UV‐Vis spectra acquisition. Many degradation compounds were identified through their exact mass (2 ppm accuracy) and MS‐MS technique. In particular, all CV demethylated products, demethylated Michler's ketone and particularly some compounds that most likely contain oxygen, such as N‐oxides, were found. Fragmentation products are all justified by the proposed fragmentation scheme, in term of precursor exact mass and isotopic profile, characteristic losses in fragmentation and rebuilt structure formula. In particular, we hypothesized the presence of N‐imido oxides and hydroxylamine derivates, never reported before, together with the demethylated derivatives of the studied dyes. All these compounds, although at trace level in our samples, contribute to the discoloration and fading of works of arts made with CV. In particular, demethylation of CV by UV light leads to formation of compounds absorbing at shorter wavelengths than CV (blue shift) or no‐absorbing in visible range (yellow‐colourless) with an overall effect that may appear reddish‐brown. This phenomenon justifies drawings appearing grey or brown on aged yellowed paper, when CV‐based inks or paints were used. The final aim was to better characterize the photo‐degradation of early synthetic dyes (in particular of CV) and to gain a better insight into the discoloration and fading of purple ink strokes made of CV. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC‐MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at ?80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC‐MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

Determination of avoparcin in animal tissues and milk using LC‐ESI‐MS/MS and tandem‐SPE

A highly sensitive and selective method using LC‐ESI‐MS/MS and tandem‐SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H]³⁺ and the major product ions of AV‐α and ‐β at m/z 637 → 86/113/130 and m/z 649 → 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem‐SPE with an ion‐exchange (SAX) and InertSep C18‐A cartridge clean‐up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV‐α (r >0.996) and ‐β (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).     

Simultaneous determination of 103 pesticide residues in tea samples by LC‐MS/MS

A simple and sensitive method was developed and validated for the simultaneous determination of 103 pesticide residues in tea by LC‐MS/MS. For the analysis of the pesticide with polarity, thermal lability or low volatility, this LC‐MS/MS method has an advantage over GC. In this work, residual pesticides were extracted from the tea sample with ACN and then purified using Carb‐NH₂ SPE cartridges. Using the multiple reaction monitoring mode, the pesticides were quantified and identified by the most abundant and characteristic fragment ions. The recoveries obtained for each pesticide ranged between 65 and 114% at three spiked concentration levels. The intra‐day precisions were lower than 19.6%. Good linear relationships were observed with the correlation coefficients r² >0.996 for all analytes. The established method was successfully applied to the determination of pesticide residues in real tea samples.     

Quantification and validation of HPLC‐UV and LC‐MS assays for therapeutic drug monitoring of ertapenem in human plasma

Rapid and simple HPLC‐UV and LC‐MS methods were developed and validated for the quantification of ertapenem (Invanz?) in human plasma. Ertapenem is a unique drug in that current dosing recommendations call for a 1 g dose for normal renal function patients, despite body weight. These assays, which involve a protein precipitation followed by liquid–liquid extraction, allow for fast therapeutic drug monitoring of ertapenem in patients, which is especially useful in special populations. Both methods were sufficient to baseline resolve meropenem (internal standard) and ertapenem, and were validated over 3 days using a six‐point calibration curve (0.5–50 µg/mL). Validation was collected using four different points on the calibrations curve yielding acceptable precision (<15% inter‐day and intra‐day; <20% for lower limit of quantitation, LLOQ) as well as accuracy (<15% inter‐day and intra‐day; <20% for LLOQ). The lower limit of detection (LOD) was determined to be 0.1 and 0.05 µg/mL for the HPLC‐UV and LC‐MS methods, respectively. The developed HPLC‐UV and LC‐MS methods for ertapenem quantification are fast, accurate and reproducible over the calibration range and can be used to determine ertapenem plasma concentrations for monitoring clinical efficacy. Copyright © 2012 John Wiley & Sons, Ltd.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed‐phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well‐suited geometries/dimensions. Here, a heart‐cut nano‐LC–CZE–MS setup was developed utilizing for the first time a mechanical 4‐port valve as LC–CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart‐cut transfer of individual LC peaks and subsequent CZE–MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE–MS. In addition, this study represents the first application of an LC–CE–MS system for intact protein analysis. The nano‐LC–CZE–MS system is expected to be applicable to various other analytical challenges.     

Comparison of LC‐UV and LC–MS methods for simultaneous determination of teriflunomide,dimethyl fumarate and fampridine in human plasma: application to rat pharmacokinetic study

This study describes a comparison between LC‐UV and LC–MS method for the simultaneous analyses of a few disease‐modifying agents of multiple sclerosis. Quantitative determination of fampridine (FAM), teriflunomide (TFM) and dimethyl fumarate (DMF) was performed in human plasma with the recovery values in the range of 85–115%. A reversed‐phase high‐performance liquid chromatography (HPLC) with UV as well as MS detection is used. The method utilizes an XBridge C₁₈ silica column and a gradient elution with mobile phase consisting of ammonium formate and acetonitrile at a flow rate of 0.5 mL min^?1. The method adequately resolves FAM, TFM and DMF within a run time of 15 min. Owing to low molecular weights, the estimation of DMF and FAM is more versatile in UV than MS detection. With LC‐UV, the detection limits of FAM, TFM and DMF were 0.1, 0.05, 0.05 μg and the quantification limit for all the analytes was 1 μg. With LC–MS, the detection and quantification limits for all of the analytes were 1 and 5 ng, respectively. The two techniques were completely validated and shown to be reproducible and sensitive. They were applied to a pharmacokinetic study in rats by a single oral dose. Copyright © 2016 John Wiley & Sons, Ltd.     

Use of high‐pH (basic/alkaline) mobile phases for LC–MS or LC–MS/MS bioanalysis

High‐pH or basic/alkaline mobile phases are not commonly used in LC–MS or LC–MS/MS bioanalysis because of the deeply rooted concern with column instability and reduced detection sensitivity for basic compounds in high‐pH mobile phases owing to charge neutralization. With the advancement of LC column technology and the wide recognition of the “wrong‐way‐round” phenomena, high‐pH mobile phases are more and more used in LC–MS or LC–MS/MS bioanalysis to improve chromatographic peak shape, retention, selectivity, resolution, and detection sensitivity, not only for basic compounds, but also for many other compounds. In this article, the benefits, practical considerations, application examples and cautions for using high‐pH mobile phases in LC–MS or LC–MS/MS bioanalysis are reviewed, with a focus on quantification. Furthermore, the future trends in this field are also envisaged. A total of 84 references are cited in this review.     

Multi‐mycotoxin analysis in complex biological matrices using LC‐ESI/MS: Experimental study using triple stage quadrupole and LTQ‐Orbitrap

In the present study, we report the application of LC‐MS based on two different LC‐MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC‐MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC‐ESI‐MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra‐assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro‐LC‐LTQ‐Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro‐LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC‐MS

A method based on pressurized liquid extraction and LC‐MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80°C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC‐MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995–0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC‐MS/MS provides a sensitive and selective method for the determination of BUs in food.     

Fractionation‐dependent improvements in proteome resolution in the mouse hippocampus by IEF LC‐MS/MS

An assessment of fractionated mouse hippocampal peptides was conducted. Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by IEF. Aliquots of fractions were pooled into fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to LC‐MS/MS analysis. Samples consisting of many individual fractions had more protein identifications, greater protein sequence coverage, and quantified proteins with more spectral counts than protein extract that was unfractionated or pooled into fewer LC‐MS/MS samples. Additionally, prefractionation reduced the median CV for spectral counts as much as 33%. However, the relative gain in proteome resolution was found to saturate with increasing fractionation extent. This study demonstrates how prefractionation by offline IEF can improve the resolution of proteomic investigations of the mouse hippocampus, and that a data‐driven pooling methodology can reduce excessive and cost‐ineffective fractionation.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.