Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample

Methanol–chloroform based protein precipitation is an essential step in many liquid chromatography–tandem mass spectrometry‐based cellular proteomics applications. However, re‐solubilization of the total protein precipitate is difficult using regular in‐solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re‐solubilization of membrane fractions. In this study, we demonstrated an application combining methanol–chloroform based protein precipitations and deoxycholic acid assisted re‐solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry‐based bottom‐up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in‐solution trypsin digestion studies were presented on methanol–chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid‐treated samples compared to other established methods. Furthermore, liquid chromatography–tandem mass spectrometry analysis of an equal amount of proteins from methanol–chloroform precipitated, and methanol–chloroform/deoxycholic acid‐treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.     

Simultaneous determination of phenolic compounds in Equisetum palustre L. by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid‐phase dispersion extraction

A method based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with tandem mass spectrometry is presented for the extraction and determination of phenolic compounds in Equisetum palustre. This method combines the high efficiency of matrix solid‐phase dispersion extraction and the rapidity, sensitivity, and accuracy of ultra high performance liquid chromatography with tandem mass spectrometry. The influential parameters of the matrix solid‐phase dispersion extraction were investigated and optimized. The optimized conditions were as follows: silica gel was selected as dispersing sorbent, the ratio of silica gel to sample was selected to be 2:1 (400/200 mg), and 8 mL of 80% methanol was used as elution solvent. Furthermore, a fast and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the determination of nine phenolic compounds in E. palustre. This method was carried out within <6 min, and exhibited satisfactory linearity, precision, and recovery. Compared with ultrasound‐assisted extraction, the proposed matrix solid‐phase dispersion procedure possessed higher extraction efficiency, and was more convenient and time saving with reduced requirements on sample and solvent amounts. All these results suggest that the developed method represents an excellent alternative for the extraction and determination of active components in plant matrices.     

Separation and purification of two minor typical diarrhetic shellfish poisoning toxins from harmful marine microalgae via combined liquid chromatography with mass spectrometric detection

A novel method was developed for the purification of two typical diarrhetic shellfish poisoning toxins from toxin‐producing marine microalgae using macroporous resin, high‐speed countercurrent chromatography–mass spectrometry, and semipreparative high‐performance liquid chromatography–mass spectrometry. Analytical high‐performance liquid chromatography–mass spectrometry was used for identification and purity analysis of okadaic acid and dinophysistoxin‐1 because they exhibit no visible or ultraviolet absorption. First, four kinds of macroporous resins were investigated, and HP‐20 macroporous resin was selected for the preenrichment and cleanup of the two target toxins. Second, the resin‐purified sample was further purified using high‐speed countercurrent chromatography coupled with a mass spectrometer. The purities of the obtained okadaic acid and dinophysistoxin‐1 were 89.0 and 83.0%, respectively, as determined through analytical high‐performance liquid chromatography–mass spectrometry. Finally, further purification was carried out using semipreparative high‐performance liquid chromatography with mass spectrometry, and the purities of the final okadaic acid and dinophysistoxin‐1 products were both over 98.0% based on the analytical high‐performance liquid chromatography–mass spectrometry chromatograms and fraction spectra. This work demonstrates that the proposed purification process is a powerful method for the preparation of high‐purity okadaic acid and dinophysistoxin‐1 from toxin‐producing marine microalgae. Moreover, it is particularly important for the purification and preparation of minor toxins that exhibit no visible or ultraviolet absorption from harmful marine algae.     

Covalently bonded aptamer‐functionalised magnetic mesoporous carbon for high‐efficiency chloramphenicol detection

A novel aptamer‐modified magnetic mesoporous carbon was prepared to develop a specific and sensitive magnetic solid‐phase extraction method through combination with ultra‐high performance liquid chromatography‐tandem mass spectrometry for the analysis chloramphenicol in complex samples. More specifically, the chloramphenicol aptamer‐modified Mg/Al layered double hydroxide magnetic mesoporous carbon was employed as a novel magnetic solid‐phase extraction sorbent for analyte enrichment and sample clean‐up. The extraction solvent, extraction time, desorption solvent, and desorption time were investigated. It was found that the mesoporous structure and aptamer‐based affinity interactions resulted in acceptable selective recognition and a good chemical stability toward trace amounts of chloramphenicol. Upon combination with the ultra‐high performance liquid chromatography‐tandem mass spectrometry technique, a specific and sensitive recognition method was developed with a low limit of detection (0.94 pmol/L, S/N = 3) for chloramphenicol analysis. The developed method was successfully employed for the determination of chloramphenicol in complex serum, milk powders, fish and chicken samples, giving recoveries of 87.0‐107% with relative standard deviations of 3.1‐9.7%.     

Determination of chemotherapeutic agents in fish and shellfish by matrix solid‐phase dispersion and liquid chromatography‐tandem mass spectrometry

Chemicals are widely used in aquaculture and one of the main recipients of these analytes is the aquatic environment. The aim of this work was to develop and validate a simple and sensitive method for the determination of multiclass chemotherapeutic agents in farmed fish and shellfish using matrix solid‐phase dispersion and liquid chromatography‐tandem mass spectrometry. Residues of azamethiphos, three avermectins, two carbamates, and two benzoylureas were extracted from samples using silica gel as clean‐up adsorbent and 0.5% acetic acid in acetonitrile as elution solvent. The extraction conditions were investigated and optimized using an experimental design. Mass spectrometry detection was carried out in positive electrospray ionization mode with multiple‐reaction monitoring scan (except for benzoylurea family). Matrix‐matched standards were used for the drugs quantification. Good linearity (R² ≥ 0.996) was observed in the range of 5–500 μg kg^?1. Limits of detection were in the range of 1.5–3.7 μg kg^?1. Recoveries from salmon samples spiked with veterinary drugs were in the range 84.9–118%. Precision was satisfactory since relative standard deviations were lower than 10.6%. The method can be successfully applied for the analysis of fish and shellfish from aquaculture.     

Comparison of pulse glow discharge‐ion mobility spectrometry and liquid chromatography with tandem mass spectrometry based on multiplug filtration cleanup for the analysis of tricaine mesylate residues in fish and water

Analytical methods based on multiplug filtration cleanup coupled with pulse glow discharge‐ion mobility spectrometry and liquid chromatography tandem mass spectrometry were developed for the analysis of tricaine mesylate residue in fish and fish‐raising water samples. A silica fiber holder and an appropriate new interface were designed to make the direct introduction of the fiber into the pulse glow discharge‐ion mobility spectrometry introduction mechanism. The multiplug filtration cleanup method with adsorption mixtures was optimized for the determination of tricaine mesylate in fish samples. Good linear relationships were obtained by the two methods. For fish samples, limits of detection were 6 and 0.6 μg/kg by ion mobility spectrometry and liquid chromatography with tandem mass spectrometry, respectively. The matrix effect of the established liquid chromatography tandem mass spectrometry method was negligible for fish samples but that of the ion mobility spectrometry method was not. The two methods were compared. The ion mobility spectrometry system could be used a rapid screening tool on site with the advantage of rapidity, simplicity, and portability, and the liquid chromatography tandem mass spectrometry system could be used for validation in laboratory conditions with the advantage of lower limit of detection, stability, and precision.     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

Residue measurement of pendimethalin in tobacco by using heart‐cutting two dimensional liquid chromatography coupled with tandem mass spectrometry

A novel heart‐cutting two‐dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for quantitative analysis of pendimethalin residue in tobacco. The strategy of reversed phase liquid chromatography coupled with another reversed‐phase liquid chromatography was employed for high column efficiency and excellent compatibility of mobile phase. In the first dimensional chromatography, a cyano column with methanol/water as the eluent was applied to separate pendimethalin from thousands of interference components in tobacco. By heart‐cutting technique, which effectively removed interference components, the target compound was cut to the second dimensional C18 column for further separation. The pendimethalin residue was finally determined by the tandem mass spectrometry under multiple reaction monitoring reversed‐phase liquid chromatography mode. Sample pretreatment of the new method was simplified, involving only extraction and filtration. Compared with traditional methodologies, the new method showed fairly high selectivity and sensitivity with almost no matrix interference. The limit of quantitation for pendimethalin was 1.21 ng/mL, whereas the overall recoveries ranged from 95.7 to 103.3%. The new method has been successfully applied to non‐stop measure of 200 real samples, without contamination of ion source. Detection results of the samples agreed well with standard method.     

Novel strategy for the facile enrichment of isopentenyl pyrophosphate in rat plasma via Ti4+‐immobilized polydopamine@Fe3O4 core–shell microspheres

In this paper, a facile extraction strategy is reported for the analysis of isopentenyl pyrophosphate, a key isoprenoid, based on magnetic core–shell microspheres with Ti⁴⁺ ion exterior walls coupled with liquid chromatography and tandem mass spectrometry. Because of their excellent hydrophilicity and biological compatibility, the polydopamine@Fe₃O₄‐Ti⁴⁺ microspheres display ideal isopentenyl pyrophosphate extraction efficiency. The technique includes three steps: sample loading, nonphosphate washing, and phosphate elution. Moreover, the microspheres can be regenerated by thorough washing with a specific solvent and can be reused multiple times. The liquid chromatography with tandem mass spectrometry separation was performed on a Welch Ultimate® XB‐C₁₈ column with a total chromatographic analysis time of 5 min; the analytical recovery was 98.52%. The proposed method was used to determine the isopentenyl pyrophosphate concentration in rat plasma samples collected from the Shanghai Chest Hospital. The results indicate the prospective value of the as‐made microspheres for the sensitive and selective enrichment of phosphate compounds in complicated matrices.     

Aspects of trapping efficiency and matrix effects in the development of a restricted‐access‐media‐based trap‐and‐elute liquid chromatography with mass spectrometry method

Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time‐demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.     

l‐Cysteine‐modified magnetic microspheres for extraction and quantification of saxitoxin in rat plasma with liquid chromatography and tandem mass spectrometry

Saxitoxin, which is one of the most typical paralytic shellfish poisoning toxins, ranks the highest intoxication rate of marine biological poisoning cases globally. Efficient clean‐up and extraction of saxitoxin from complex biological matrices are imperative for the analysis and concentration monitoring of the toxin when correlative poisoning cases happen. Herein, l ‐cysteine‐modified magnetic microspheres based on metal‐organic coordination were synthesized by a facile approach and applied for magnetic solid‐phase extraction of saxitoxin from rat plasma samples before liquid chromatography–tandem mass spectrometry detection. Parameters, including adsorbent amount, extraction time, desorption solution, and desorption time that could affect the extraction efficiency, were respectively investigated. The developed method demonstrated good linearity in the range of 5–300 ng/mL (R² = 0.9985) with a limit of quantification of 5 ng/mL and a limit of detection of 0.5 ng/mL, acceptable accuracy. and precision of within‐run and between‐run.     

Determination of Synacthen in urine for sports drug testing by means of nano‐ultra‐performance liquid chromatography/tandem mass spectrometry

Doping control analysis of performance‐enhancing peptides in urine represents a challenging requirement in modern sports drug testing. Low dosing, effective metabolism and short half‐life lead to target concentrations in the low fmol/mL range in urine. Synthetic adrenocorticotropic hormone (1‐24, Syn‐ACTH‐en) shares all these characteristics and improved analytical performance is required for its sufficient determination by means of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The desired effects for cheating sportsmen are mainly due to enhanced release of corticosteroids as well as androgenic steroids into the circulation after systemic administration of the drug. Immunoaffinity purification with coated magnetic beads and subsequent liquid chromatography with nano‐ultra‐performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (high resolution/high mass accuracy) of Synacthen from urinary specimens is described in the present study. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (3 pg/mL), linearity, precision (<20%), recovery (～30%), robustness, specificity and stability. For all experiments, the ACTH fragment 1‐17 was used as the internal standard. Copyright © 2009 John Wiley & Sons, Ltd.     

Determining multi‐pesticide residues in teas by dispersive solid‐phase extraction combined with speed‐regulated directly suspended droplet microextraction followed by gas chromatography–tandem mass spectrometry

In this study, an effective speed‐regulated directly suspended droplet microextraction method was developed to condense pesticide residues from teas through dispersive solid‐phase extraction prior to analysis by gas chromatography with tandem mass spectrometry. The extractant was intentionally dispersed into the sample solution in the form of globules through high‐speed agitation. This procedure increases the contact area between the binary phases and shortens the distribution equilibrium time. The fine globules reassembled by decelerating stirring speed, the extractant could be taken out for gas chromatography with tandem mass spectrometry. Recovery studies were performed under optimized extraction conditions by using matrix blanks fortified with pesticides at three concentrations (10, 50, and 100 µg/kg). Over 87% of the recoveries for the analytes in four tea matrices were acceptable given their recovery ranges of 70–120% and relative standard deviations of ≤20%. The limits of quantification of most pesticides were lower than 10 µg/kg and thus satisfied the requirements for maximum residue levels prescribed by the European Community. A total of 38 tea samples from local markets were analyzed by using the proposed method. Results showed that chlorpyrifos was the most frequently detected pesticide in teas. The method is a potential choice for the routine monitoring of pesticide residues in complex matrices.     

The development and validation of a high‐throughput LC–MS/MS method for the analysis of endogenous β‐hydroxy‐β‐methylbutyrate in human plasma

A high‐throughput, sensitive, and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid quantitation of β ‐hydroxy‐β ‐methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC–MS/MS technique under negative mode electrospray ionization conditions. A ¹³C–labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze–thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter‐day accuracies and coefficients of variation (CV) were 91.2–98.1 and 3.7–7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers.     

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

A highly sensitive and simple diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high‐performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90–105% in tap water and 94–97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Self‐acidity induced effervescence and manual shaking‐assisted microextraction of neonicotinoid insecticides in orange juice

A novel dispersive liquid‐liquid microextraction that combines self‐induced acid‐base effervescent reaction and manual shaking, coupled with ultra high performance liquid chromatography with tandem mass spectrometry was developed for simultaneous determination of ten neonicotinoid insecticides and metabolites in orange juice. An innovative aspect of this method was the utilization of the acidity of the juice for a self‐reaction between acidic components contained in the juice sample and added sodium carbonate which generated carbon dioxide bubbles in situ, accelerating the analytes transfer to the extractant of 1‐undecanol. The total acid content of juice sample was measured to produce the maximum amount of bubbles with minimum usage of carbonate. Manual shaking was subsequently adopted and was proven to enhance the extraction efficiency. The factors affecting the performance, including the type and the amount of the carbon dioxide source and extractant, and ionic strength were optimized. Compared with conventional methods, this approach exhibited low limits of detection (0.001–0.1 µg/L), good recoveries (86.2–103.6%), high enrichment factors (25–50), and negligible matrix effects (?12.3–13.7%). The proposed method was demonstrated to provide a rapid, practical, and environmentally friendly procedure due to no acid reagent, toxic solvent, or external energy requirement, giving rise to potential application on other high acid‐content matrices.     

Comparative study of matrices for their use in the rapid screening of anabolic steroids by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

New data on sample preparation and matrix selection for the fast screening of androgenic anabolic steroids (AAS) by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) is presented. The rapid screening of 15 steroids included in the World Anti‐Doping Agency (WADA) prohibited list using MALDI was evaluated. Nine organic and two inorganic matrices were assessed in order to determine the best matrix for steroid identification in terms of ionisation yield and interference by characteristic matrix ions. The best results were achieved for the organic matrices 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) and trans‐3‐indoleacrylic acid (IAA). Good signals for all the steroids studied were obtained for concentrations as low as 0.010 and 0.050 µg/mL on the MALDI sample plate for the HABA and IAA matrices, respectively. For these two matrices, the sensitivity achieved by MALDI is comparable with the sensitivity achieved by gas chromatography/mass spectrometry (GC/MS), which is the conventional technique used for AAS detection. Furthermore, the accuracy and precision obtained with MALDI are very good, since an internal mass calibration is performed with the matrix ions. For the inorganic matrices, laser fluences higher than those used with organic matrices are required to obtain good MALDI signals. When inorganic matrices were used in combination with glycerol as a dispersing agent, an important reduction of the background noise was observed. Urine samples spiked with the study compounds were processed by solid‐phase extraction (SPE) and the screening was consistently positive. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis

An accurate and precise method for the quantification of 11‐nor‐Δ⁹‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for doping analysis purposes has been developed. The method involves the use of only 200 µL of urine and the use of D₉‐THCA as internal standard. No extraction procedure is used. The urine samples are hydrolysed using sodium hydroxide and diluted with a mixture of methanol/glacial acetic acid (1:1). Chromatographic separation is achieved using a C8 column with gradient elution. All MS and MS/MS parameters were optimised in both positive and negative electrospray ionisation modes. For the identification and the quantification of THCA three product ions are monitored in both ionisation modes. The method is linear over the studied range (5–40 ng/mL), with satisfactory intra‐and inter‐assay precision, and the relative standard deviations (RSDs) are lower than 15%. Good accuracy is achieved with bias less than 10% at all levels tested. No significant matrix effects are observed. The selectivity and specificity are satisfactory, and no interferences are detected. The LC/MS/MS method was applied for the analysis of 48 real urine samples previously analysed with a routine gas chromatography/mass spectrometry (GC/MS) method. A good correlation between the two methods was obtained (r² > 0.98) with a slope close to 1. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.