Elution Speed Research Articles

Determination of oxylipins and their precursors in breast milk by solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry

Oxylipins and their precursors (long-chain polyunsaturated fatty acids, LCPUFAs) are key intercellular signaling molecules influencing the inflammatory response. Each oxylipin has pro- and/or anti-inflammatory effects, and the relative abundance of different oxylipins can alter the inflammatory balance, making it important to clarify the oxylipin profile of breast milk for optimal infant health. The extraction, identification, and simultaneous quantification of oxylipins in breast milk are challenging due to the structural similarity, limited stability, and the low endogenous concentration of oxylipins and the complex matrix of breast milk. This study aimed to develop a solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (SPE-UPLC-MS/MS) method for the comprehensive and specific quantification of oxylipins and their precursors in breast milk. The LC conditions (including column, mobile phase, and gradient conditions) and SPE procedure (including SPE cartridges, elution solvent, and elution volume) were optimized to achieve accurate quantification and better analyte recovery. A single 18-minute chromatographic run allows for the quantification of 20 oxylipins and 5 PUFAs. The results showed good linearity (R2 > 0.99) over the concentration range of 2 to 100 ng/mL, with the instrument detection limits ranging from 0.01 to 0.90 ng/mL for oxylipins and 0.02 to 0.59 ng/mL for PUFAs. The method is rapid, sensitive, and reproducible (RSD ≤ 10%) and is suitable for the quantitative analysis of oxylipins and their precursors in infant formula samples.

Quantitative and qualitative comparison of marine dissolved organic nitrogen recovery using solid phase extraction

AbstractMarine dissolved organic carbon and nitrogen (DOC and DON) are major global carbon and nutrient reservoirs, and their characterization relies on extraction methods for preconcentration and salt removal. Existing methods optimize for capturing and describing DOC. Here, we report an optimized analytical strategy to recover marine DON for subsequent molecular characterization. Retention efficiencies between 5% and 95% are reported for seven solid phase extraction (SPE) sorbents, with PPL recovering 23% of marine DON compared to 95% recovered with C18. Additional comparisons of the effect of varying sample volumes and elution speed, and the resulting molecular composition of DON extracts, were investigated using C18 and PPL sorbents. Sample volumes > 200 mL decreased DON retention efficiency independent of SPE sorbent, and gravity elution recovered 1.7‐ to 4.2‐fold more DON compared to vacuum elution. Characterization of extracted DON by negative‐ion electrospray ionization Fourier transform‐ion cyclotron resonance mass spectrometry (FT‐ICR MS) highlights compositional differences between DON species recovered by each method. DON isolated with optimized methods includes low molecular weight (< 600 Da) peptide‐like compounds with low O:C ratios (0.2 to 0.5) that are not detected by other SPE sorbents (e.g., PPL). The majority of additional DON isolated with this approach was undetectable by direct infusion negative mode FT‐ICR MS analysis.

Coconut shell biochar application in liquid-solid microextraction of triazine herbicides from multi-media environmental samples

A rapid, fast, widely applicable liquid-solid microextraction and purification method of triazine herbicides (TRZHs) in muti-media samples using salting-out assisted liquid-liquid extraction (SALLE) combined with self-assembled monolithic spin columns-solid phase micro extraction (MSC-SPME) was developed. Environmentally friendly coconut shell biochar (CSB) was used as the adsorbents of MSC-SPME. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was the separation and determination method. The adsorption kinetics and isotherms were investigated to indicate the interaction between CSB and TRZHs. Several parameters influencing the liquid-solid microextraction efficiency, such as sample pH, salting-out solution volume and pH, sample loading speed, elution speed, elution ratio and volume of eluent were systematically investigated with the aid of orthogonal design. The whole extraction process was operated within 10 min. Under the optimum extraction and determination conditions, good linearities for three TRZHs were obtained in a range of 0.10–200.00 ng mL−1, with linear coefficients (R2) greater than 0.999. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 6.99–11.00 ng L−1 and 23.33–36.68 ng L−1, respectively. The recoveries of the three TRZHs in multi-media environmental samples were ranged from 69.00% to 124.72%, with relative standard deviations (RSDs) lower than 0.43%. This SALLE-MSC-SPME-UPLC-MS/MS method was successfully applied to the determination of TRZHs in environmental and food samples and exhibited the advantages of high efficiency and sensitivity, low cost, and environmental friendliness. Compared with the methods published before, CSB-MSC was green, rapid, easy-operated, and reduced the whole cost of the experiment; SALLE combined MSC-SPME eliminated the matrix references effectively; what's more, the SALLE-MSC-SPME-UPLC-MS/MS method could be applied to various sample without complicated sample pretreatment procedure.

The effect of elution speed control on purity of separated large-diameter single-walled carbon nanotubes in gel chromatography

Large-diameter single-walled carbon nanotubes (SWNTs) have unique properties compared to small-diameter ones, for example, higher electrical conductivities, higher charge carrier mobilities, smaller bandgaps, and longer lengths, which can broaden SWNT applications. However, because the preparation methods result in mixtures of metallic SWNTs (m-SWNTs) and semiconducting SWNTs (s-SWNTs); such as in the case of small-diameter SWNTs, they must be separated and used for specific applications to obtain optimal performance characteristics. In this study, we show that simple elution speed control can increase the purity of separated large-diameter SWNTs in gel chromatography. According to the results, the flow rate of the eluant (i.e., the elution speed during gel column chromatography) greatly affects the purity of the separated m-SWNTs. We controlled the elution speed by changing the pressure inside the column during the separation process and experimentally investigated the relationship between the purity of the separated m-SWNTs and elution speed. By adjusting the elution speed to 0.13 ml/min, we obtained large quantities of pure m-SWNTs. Moreover, high-purity s-SWNTs were obtained for a wide elution speed range. Thus, highly pure m- and s-SWNTs can simultaneously be prepared by simply controlling the elution speed in single-column gel chromatography.

HPLC–MS/MS Analysis and Study on the Adsorption/Desorption Characteristics of Ginsenosides on Anion-Exchange Macroporous Resins

Anion exchange resin is a modified version of polar/nonpolar macro porous resins with enhanced selectivity on compounds carrying negative charges. Although it is a promising adsorbent for the purification of natural products, no research regarding its application on ginsenosides has been reported elsewhere. In this paper, we first isolated total ginsenosides from Panax ginseng and investigated their static adsorption/desorption capacity on different resins, among which D301 resin was selected as the optimum one. A pH value of 8 was chosen for the balanced interaction between total ginsenosides and the adsorbents. Adsorption kinetics and isotherms of total ginsenosides on D301 and D101 resins were investigated to differentiate the adsorption characteristics of anion exchange and nonpolar macroporous resins. A D301-based chromatographic method was established to purify total ginsenosides with settings as follows: sample loading and elution speed = 4 bed volume per hour, breakthrough volume = 2 bed volume, elution solvent = 80% aqueous ethanol, elution volume = 8 bed volume. HPLC-QqQ-MS qualitatively and quantitatively analyzed nine individual ginsenosides from the eluates, demonstrating an enrichment factor of 5.3 as well as a recovery rate of 80.9% for the whole of these ginsenosides. The purity of these ginsenosides in Panax ginseng extract increased from 17.07% to 91.19% after the purification. Hence, anion exchange resin D301 proved to be a more comprehensive and efficient adsorbent than the conventional nonpolar macroporous resin for the separation of total ginsenosides from natural sources. Based on the findings in this paper, the enrichment process for total ginsenosides could be well established via one-step column chromatography.

Liver and kidney serum profile abnormalities in workers exposed to aflatoxin B1 in urban solid waste management centers.

Many workers are exposed to health problems arising from molds, fungi, and their toxins during waste processing. Aflatoxin B1 (AFB1) level in airborne and settled dust, aflatoxin B1-albumin (AFB1-Alb) adduct in serum, liver and kidney biochemical tests, and body redox change of workers in municipal dry waste-processing sites were investigated. The surface, personal, and area air dust and the blood of workers' samples were collected from the plastic and bread waste-sorting sections in three recycling municipal dry waste sites. Digestion (only for serum samples), passed through SPE cartridge, elution, and collection with methanol, immune-affinity column clean-up, and HPLC system equipped with post-column derivatization method and fluorescence detection were performed for determination of AFB1 and AFB1-Alb levels in the samples. The mean level of dust and AFB1 in the personal and area air, and in the settled dust and the AFB1-Alb in the serum of workers in the bread waste sorting, was higher than plastic waste-sorting samples, in all of the sites. The differences in the biochemical profiles of subjects exposed to aflatoxin B1 as compared to the control group especially in liver and kidney function parameters as well as antioxidant factors of the serum were significant. The workers in handling of municipal waste may be exposed to potentially hazardous levels of aflatoxin B1. The adverse effects of AFB1 on the kidney and liver may be caused by changes in the redox system.

Separation of Heavy Lanthanoids by Flash Column Chromatography for Precise Determination of Er and Yb Isotope Compositions in Rock Samples

We present a new column chemistry technique for the quantitative separation of heavy lanthanoids by an ultra‐fine‐grained LN resin (20–50 µm) with a specific emphasis on the purification of Er and Yb for their isotopic analysis. To achieve the quantitative separation of Er and Yb within a reasonable timescale, flash column chromatography was applied, where the column was attached to a newly designed vacuum box system, thus accelerating the elution speed by ten times compared with that of the normal column procedure operated by gravity flow. The recovery yields of Er and Yb were confirmed to be approximately 100%, which is important to suppress the effect of the mass‐independent fractionation of the Er and Yb isotopes during chromatography. Additionally, we have developed precise Er and Yb isotope measurements by thermal ionisation mass spectrometry (TIMS) using multistatic and/or dynamic methods. Moreover, in most cases, the Er and Yb isotope compositions of the measured four terrestrial rock samples were indistinguishable from those of the commercially available Er and Yb Alfa Aesar solutions. The new method presented in this work will be useful for future studies on heavy lanthanoids in various geological materials.

Research on method of gravity thermal coupling field-flow fractionation based on symmetric encryption algorithm

The most frequently used method includes the gravity field-flow separation and thermal field-flow separation. By analyzing the influence of velocity of carrier flow on the separation effect of gravity field flow, we can know that the movement track of particle in different carrier liquid elution speed was significantly different when the external carrier liquid was continuously injected into the gravity field-flow separation channel, so the additional lift force generated by the thermal field was used to enhance the driving lift force of particles in carrier liquid and the effect of field-flow separation, and thus to achieve the gravity thermal coupling field-flow separation. In order to ensure the accuracy of field-flow separation, the computer vision detection system was used to detect the separation effect of gravity thermal coupling field flow. In this process, the symmetrical encryption optimization algorithm based on 3D cat mapping was used to realize image encryption through image scrambling and image mixing, so as to ensure the integrity of image. Experimental results show that the gravity thermal-coupled field-flow separation method based on symmetric encryption algorithm can separate the starch particles with different sizes effectively, which is suitable for the purification of powdery particles.

Enhancement in the computation of gradient retention times in liquid chromatography using root-finding methods

Gradient elution may provide adequate separations within acceptably short times in a single run, by gradually increasing the elution speed. Similarly to isocratic elution, chromatograms can be predicted under any experimental condition, through strategies based on retention models. The most usual approach implies solving an integral equation (i.e., the fundamental equation of gradient elution), which has an analytical solution only for certain combinations of retention model and gradient programme. This limitation can be overcome by using numerical integration, which is a universal approach although at the cost of longer computation times. In this work, several alternatives to improve the performance in the resolution of the integral equation are explored, which can be especially useful with multi-linear gradients. For this purpose, the application of several root-finding methods that include the Newton's and bisection searches is explored in three frameworks: isolated predictions, regression modelling problems using gradient training sets, and optimisation of multi-linear gradients. Significant reductions of computation times were obtained. The substitution of non-integrable retention models by Tchebyshev polynomial approximations, which are pre-calculated before solving the integral equation in optimisation problems, is also investigated.

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry.

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

Batch and fixed bed column studies on phenolic adsorption from wine vinasses by polymeric resins

The adsorption of phenolic compounds from wine vinasses onto non ionic resins was investigated with the goal of recovering antioxidant components from the effluent. Batch studies showed that the kinetic data fit to a pseudo-second order kinetic model. Dynamic adsorption studies with macroporous resins packed columns were also conducted to analyze the effect of initial phenolic concentration, the elution speed and the length of the packed bed on breakthrough curves. The experimental data showed that with increased column bed depth from 10 to 20 cm, breakpoint time (tb) increased from 19.6 to 25.6 min. The breakthrough curve was simulated well by Bohart-Adams, Yoon-Nelson and Thomas models. Bohart-Adams model was the only effective to fit the initial part (1–t1/2). The maximum removal capacity (qTh) calculated by Thomas model reached 98.3 mg/g at a flow rate of 5 mL/min.

Purification of high-purity glycyrrhizin from licorice using hydrophilic interaction solid phase extraction coupled with preparative reversed-phase liquid chromatography.

Glycyrrhizin (GA), a major bioactive compound in licorice, has been extensively used throughout the world as a medicine to treat chronic viral hepatitis and allergic dermatitis. In this study, a new method based on hydrophilic interaction solid phase extraction (HILIC-SPE) and preparative reversed-phase liquid chromatography (prep-RPLC) was developed to purify GA with high purity from the complex licorice extract. Via evaluation of retention behavior of GA and flavonoids in different commercially available columns, a hydrophilic column--Click XIon was finally chosen for the purification due to its excellent resolution toward GA and flavonoids under HILIC mode. To optimize the SPE elution conditions, relative factors including water content, pH and ionic strength had been investigated in chromatographic condition. The result indicated that the most appropriate water content was 30% and pH at 4.00, as well as salt concentration should be controlled at 5mM. In addition, the optimization revealed that GA experiences both hydrophilic interaction and ion-exchange interaction on the Click XIon material. According to the chromatographic evaluation, the optimized conditions were applied to HILIC-SPE to enrich GA from licorice, which leads to an increased content of GA from 13.67% to 64.22%. Finally, prep-RPLC was performed to obtain GA with purity higher than 99.00%，which demonstrating great prospect in large-scale preparation of GA.

Determination of pesticide residues in tea by gas chromatography/triple quadrupole mass spectrometry with solid-phase extraction.

An analytical method was developed for determination of multipesticide residues, including organophosphorus, organohalogen, pyrethroid, and organonitrogen, in tea at trace levels by GC coupled with triple quadrupole mass chromatography (QqQ-MS/MS). Scan time was selected in order to optimize QqQ-MS/MS conditions. The key parameters for controlling cleanup performance were optimized, including SPE cartridge type and elution solvent volume. Acetonitrile was the extraction solvent, and a novel multilayer SPE cartridge, Cleanert TPT, was used in the cleanup step. The recoveries of the studied pesticides at 5.0, 10.0, and 25.0 microg/kg were in the range of 77.8 to 103.8% with an RSD of less than 14%. Determination coefficient (R2) values between 0.9951 and 0.9998 were obtained for all target compounds. The LOD was between 0.002 and 1.0 microg/kg, and LOQs were 0.0066-3.3 microg/kg, which satisfied the maximum residue limits for pesticides in tea recommended by the European Union and Japan. The optimized method was applied to the analysis of real tea samples obtained from the local market.

Quantitative determination of 26 steroids in eggs from various species using liquid chromatography–triple quadrupole-mass spectrometry

A method for analyzing 26 types of steroids in egg matrix was developed. The method used liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in electrospray ionization mode (ESI). The procedure involved extraction with acetonitrile and removal of phospholipids by zinc chloride (ZnCl2) followed by SPE cleanup with a Plexa cartridge. The effect of ZnCl2 on phospholipid removal was directly observed using the post column infusion procedure. The SPE washing and elution conditions were optimized using a shallow gradient procedure. The free and conjugated steroids forms were determined using enzyme hydrolysis. The developed method resulted in satisfactory precision (RSD≤15%), and the limits of quantification were between 0.05 and 25ng/g depending on the steroid types. The recoveries ranged from 63.2% to 121.5%. Finally, the developed method was successfully applied to compare the steroids in eggs from different species (i.e., hen, duck, quail and pigeon eggs) or different raising system (i.e., normal vs. organic eggs). The steroids can be clearly clustered according to species and raising system. The hierarchical clustering analysis indicated similarity of the steroids among the species. The developed method is sensitive and useful for detection and quantification of steroids in eggs and can be used for residue control programs. In addition, the observed steroid content will provide a fundamental reference for food risk assessment analysis.

Profiling analysis of biogenic amines and their acidic metabolites in mouse brain tissue using gas chromatography–tandem mass spectrometry

The profiling analysis of biogenic amines, including catecholamines and serotonin, and their metabolites in mouse brain tissue provides an important key to understanding their roles in the body and the possibility of simple and accurate diagnosis of neural diseases. A novel approach in the analysis of biogenic amines and their acidic metabolites in brain tissue using gas chromatography-tandem mass spectrometry (GC-MS/MS) is presented. Biogenic amines and their acidic metabolites in brain tissue were effectively separated using a mixed-cation-exchange solid-phase extraction (MCX-SPE) cartridge with a variation in the composition of the SPE elution solvents. A selective derivatization with hexamethyldisilazane (HMDS) and N-methyl-bis-heptafluorobutyramide (MBHFBA) was used to increase the detection sensitivity and to prevent the formation of any side-products. The identification and quantification of the target analytes were performed by gas chromatography triple quadrupole mass spectrometry (GC-MS/MS) using multiple ion reaction monitoring (MRM) mode. The overall recovery yields of the biogenic amines and their metabolites were above 87.5% at 10ng/g and 92.4% at 100ng/g of spiking concentration range. The isotopic-labeled internal standards were used for the precise quantification of bioamines and their metabolites. The calibration curves for the biogenic amines and their metabolites obtained through GC-MS/MS were linear (r(2)>0.995) over the concentration range of 1 (2 or 3)-200ng/mL. The present method was reproducible (relative standard deviation range 0.6-9.3%) and accurate (range 85.4-107.9%), with LLOQs of 0.71-3.69ng/mL. The developed method was successfully applied to the determination and quantification of bioamines and their metabolites in rat brain tissue samples.

Comprehensive profiling analysis of bioamines and their acidic metabolites in human urine by gas chromatography/mass spectrometry combined with selective derivatization

A comprehensive analytical method was developed for the profiling of biogenic amines in human urine using GC/MS in SIM mode. Biogenic amines and their acidic metabolites were converted into their volatile O-trimethylsilyl/N-heptafluorobutyryl (OTMS/-NHFBA) derivatives for GC/MS analysis. Dual hexamethyldisilazane (HMDS)/-N-methyl-bis-heptafluorobutyramide (MBHFBA) derivatizations have been shown to be quite effective, with high derivatization yields and the absence of side products for primary biogenic amines. In this study, selective derivatization conditions by HMDS/MBHFBA were optimized in terms of the reagent amount, reaction temperature and reaction time period. The highest derivatization reaction yield was obtained at 40°C for 10min for OTMS derivatization and 80°C for 5min for N-HFBA derivatization. The use of MCX SPE cartridges with different SPE elution solvents was effective for the pre-concentration and selective cleanup of the biogenic amines and their acidic metabolites in human urine. The selection of appropriate ions in SIM mode provided reliable quantification and identification and a reduction in background effects. The established method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), precision, and accuracy. The present method was linear (r2>0.996), reproducible (relative standard deviation range 1.1–6.9%), and accurate (range 87.9–111.9%), with LOQs of 0.17–17.84ng/mL. The biogenic amine profiling of human urine was successfully accomplished by GC/MS in SIM mode combined with selective HMDS/MBHFBA derivatization and MCX SPE cleanup.

Implication of Free Cholesterol in Lc–Ms Response Enhancement

In the context of matrix effects in bioanalytical LC-MS/MS, a speculative link between free cholesterol, the recoveries of the compound from three common extraction procedures, and response enhancement was qualitatively investigated. Injections on-column of cholesterol both in solution and extracts, in conjunction with post-column infusion of three representative drugs, reveal a direct role in pronounced response enhancement for two out of three analytes, under one set of LC-MS/MS conditions, for the majority of typical plasma extraction procedures, where electrospray-based gaseous ion generation is used. Cholesterol has been shown to have a strong association with LC-MS/MS response enhancement and ideally should be monitored during method development, reinforcing the reasoning behind minimizing SPE elution volumes and avoiding less selective means of sample preparation.

Advances in field-flow fractionation for the analysis of biomolecules: instrument design and hyphenation

Field-flow fractionation (FFF) separates analytes by use of an axial channel-flow and a cross-field. Its soft separation capability makes it an ideal tool for initial fractionation of complex mixtures, but large elution volumes and high flow rates have limited its applicability without significant user handling. Recent advances in instrumentation and miniaturization have successfully reduced channel size and elution speed, and thus the volume of each fraction, making it possible to conveniently couple FFF with orthogonal separation techniques for improved resolution. More detailed analysis can also be performed on the fractions generated by FFF by use of diverse analytical techniques, including MS, NMR, and even X-ray scattering. These developmental trends have given FFF more power in the analysis of different types of molecule, and will be the direction of choice for further advances in FFF technology.

Employing a new modified nanoporous carbon for extraction and determination of 1,10-phenanthroline and 2,2′-bipyridine by SPE and use of the Taguchi optimization method

Using the Taguchi method, the extraction of 1,10-phenanthroline and 2,2′-bipyridine from aqueous samples by a solid phase extraction system has been studied. The extraction is based on a new nanoporous CMK-1 modified with sodium dodecyl sulfate and Fe3+. The structural order and textural properties of the modified sorbent were studied by X-ray diffraction, nitrogen adsorption isotherm, scanning electron microscopy (SEM) and FT-IR techniques. The 1,10-phenanthroline and 2,2′-bipyridine were selectively adsorbed on this sorbent, and subsequently were recovered with dimethyl sulfoxide. The extraction efficiencies were measured by UV-Vis spectroscopy. The controllable factors such as pH, solution speed, sorbent mass, eluent volume and elution speed were optimized applying the Taguchi method. The results showed that the dynamic linear range exhibited a good linearity between 20 and 600 μg L−1. The limits of detection for 1,10-phenanthroline and 2,2′-bipyridine were 8.75 and 10.1 μg L−1 respectively and the extraction efficiencies were greater than 93% under the optimum conditions. Finally, this method was successfully applied for determination of these compounds in real samples.

Simultaneous Determination of 1,4‐Benzodiazepines and Tricyclic Antidepressants in Saliva after Sequential SPE Elution by the Same HPLC Conditions

AbstractA new method has been described to determine both benzodiazepines (six) and tricyclic antidepressants (four) simultaneously in saliva by HPLC with a UV detector set at 240 nm using cholchicine as the internal standard. A careful specific sequential solid‐phase elution was optimized and performed to elute benzodiazepines using a mixture of methanol‐acetonitrile (1:1 v/v) followed by the elution of tricyclic antidepressants with methanol. Separation of the compounds was performed on a Kromasil column (250 × 4 mm, 5 μm) by a gradient eluents consisting of 0.05 M CH3COONH4‐acetonitrile‐methanol (55:15:30 v/v/v). The results were linear for both benzodiazepines and tricyclic antidepressants up to 20 ng μL‐1 with the correlation coefficients greater than 0.998. The sensitivity limits, LOD and LOQ were 0.08‐0.34 ng μL‐1 and 0.28‐1.13 ng μL‐1, respectively. The method is simple, fast and reliable with good specificity and sensitivity, will be suitable for use in a clinical setting, where there is a concomitant use of 1,4‐benzodiazepines and tricyclic antidepressants.