Validation of a multi-analyte LC–MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair

A multi-analyte method for the detection and quantification of 87 psychoactive drugs (antidepressants, antipsychotics, benzodiazepines, and z-drugs) in human hair has been developed and fully validated using the liquid chromatography–tandem mass spectrometry system. Due to the remarkable increase in requests of hair sample tests (such as for driver’s license renewals, child custody, DFA cases, and postmortem toxicology), we focused on the development of a rapid sample preparation. About 20 mg of hair samples, previously washed and cut into snippets, was ultrasonicated with 700 μl of methanol. Samples were then directly analyzed using a 4000 QTRAP (AB SCIEX, Foster City, CA, USA) with an electrospray ionization (ESI) Turbo V^TM Ion Source. The validation criteria parameters were satisfactory and in accordance with the international guidelines. All the compounds tested were successfully detected. One important aspect is the LODs in the low picogram per milligram concentration which may suggest a potential use of this method in cases of detection of single drug exposure. However, the LC–MS/MS method has been successfully applied for the analysis of postmortem cases (n?=?9).     

Determination of Chloramphenicol in Milk Using a QuEChERS-Based on Liquid Chromatography Tandem Mass Spectrometry Method

A quantitative method for the determination of chloramphenicol in milk samples was developed based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach for liquid chromatography–tandem mass spectrometry (LC–MS/MS). Homogenized milk samples were extracted with acetonitrile. The partitioning step was performed after the addition of magnesium sulfate and sodium chloride. Chloramphenicol was determined using the electrospray negative ionization mode with tandem mass spectrometry. The procedure was validated according to the requirements of Commission Decision 2002/657/EC. The apparent recovery ranged from 90% to 110% and within-laboratory reproducibility was lower than 12%. The calculated limit of decision was 0.10 μg kg^?1 and the detection capability was 0.15 μg kg^?1. Validation results demonstrated that this method fulfills criteria for the determination of chloramphenicol in milk.     

Rapid Determination of Polar and Non-Polar Pesticides in Human Serum, Using Mixed-Mode C-C18 Monolithic Spin Column Extraction and LC–MS/MS

Organophosphates and carbamates are pesticides whose acute toxicities are caused by inhibition of acetylcholinesterase. A liquid chromatography–mass spectrometry/mass spectrometry method was developed and validated for the quantification of 16 organophosphate and carbamate insecticides in human serum. Nonpolar and polar pesticides were simultaneously extracted from serum samples using a simple and fast monolithic spin column procedure using mixed-mode C-C₁₈ cartridges. Recovery was achieved in the range 11.9–99.2 %. Chromatography was carried out on an InertSustain^® C₁₈ HP 3 μm analytical column with gradient elution. Mass spectrometric analysis was performed using an Agilent 6410 Triple Quad Tandem mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The assay was validated over a large concentration range and the limits of detection for all compounds ranged from 0.1 to 50 ng mL^?1. The precision for both intra- and inter-day determination of all analytes was found to be acceptable (< 12.9 %) and no significant matrix effect was observed. The developed method was effectively applied to clinical samples from patients presenting at hospital with symptoms of acute intentional organophosphate or carbamate poisoning, demonstrating its applicability to diagnostic applications.     

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C₁₈ column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL^?1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL^?1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.     

Optimization of Modern Analytical SPME and SPDE Methods for Determination of <Emphasis Type="Italic">Trans</Emphasis>-2-nonenal in Barley,Malt and Beer

Trans-2-nonenal is an aldehyde contributing to an unpleasant off-flavor and odor of rancid butter in stored beer. The automated solid-phase microextraction technique (SPME) coupled with gas chromatography (GC) and solid-phase dynamic extraction (SPDE) coupled with gas chromatography were optimized and introduced to determine trans-2-nonenal in barley, malt and beer. Five types of SPME fibers coated with different stationary phases (100 μm PDMS, 65 μm PDMS/DVB, 85 μm CAR/PDMS, 50/30 μm DVB/CAR/PDMS, 85 μm PA) and two needles (PDMS, PDMS/AC) were compared and tested for their efficiencies in the headspace (HS) SPME and SPDE determination of trans-2-nonenal in barley, malt and beer. The highest extraction efficiency of HS-SPME was achieved with the PDMS/DVB fiber, and addition of 1.5 g of NaCl, extraction time was 20 min at 60 °C. The highest extraction efficiency of HS-SPDE was obtained with the PDMS needle, 15 extraction strokes at 60 °C and addition of 1.5 g of NaCl. Trans-2-nonenal was identified with the method of HS-SPME coupled gas chromatography-mass spectrometry (GC–MS); the samples were analyzed using the HS-SPME-GC-coupled gas chromatography-flame ionization detector (GC-FID) technique.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Determination of Shikimic Acid in Fruits of Illicium Species and Various Other Plant Samples by LC–UV and LC–ESI–MS

A simple and specific analytical method for the quantitative determination of shikimic acid from the methanol extract of the fruits of Illicium species and from various plant samples was developed. The LC–UV separation was achieved by reversed-phase chromatography on a C18 column with potassium dihydrogen phosphate and methanol as the mobile phase. In the LC–MS method, the separation was achieved by a C12 column using water and acetonitrile, both containing 0.1% acetic acid as the mobile phase. The methods were successfully used to study the percentage compositions of shikimic acid present in nine species of Illicium and various other plant samples. The detector response was linear with concentrations of shikimic acid in the range from 1.0–500.0 μg mL^?1 by LC–UV and 100–1000 ng mL^?1 by LC–MS. Mass spectrometry coupled with electrospray ionization interface is described for the identification of shikimic acid in various plant samples. This method involved the use of the [M-H]^? ions of shikimic acid at m/z 173.0455 (calculated mass) in the negative ion mode with extractive ion monitoring.     

Validated LC–MS–MS Method for Quantitative Determination of Batifiban in Human Plasma and Its Application to a Pharmacokinetic Study

Batifiban is a new platelet GPIIb/IIIa receptor antagonist. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry has been firstly developed and validated for the quantitative measurement of batifiban in human plasma to support the investigation of this compound. Separation of analyte and the internal standard eptifibatide was performed on a Thermo HyPURITY C18 column (150 × 2.1 mm, 5 μm) with a mobile phase consisting of formic acid 0.1% (v/v)–acetonitrile (40:60, v/v) at a flow rate of 0.25 mL min^?1. The Waters QuattroMicro API triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transition m/z 819.2 → m/z (623.9 + 159.4) for batifiban and m/z 833.4 → m/z (645.7 + 159.3) for IS. The method was linear over the concentration range of 2.45–5,000 μg L^?1. The intra- and inter- day precisions were less than 15% in terms of relative standard deviation, and the accuracy was within 8.5% in terms of relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 2.45 μg L^?1 with acceptable precision and accuracy. The validated method offered sensitivity and wide linear concentration range. This method was successfully applied for the evaluation of pharmacokinetics of batifiban afer single oral doses of 55, 110 and 220 μg kg^?1 batifiban to 36 Chinese healthy volunteers.     

Rapid Determination of Polar and Non-Polar Pesticides in Human Serum,Using Mixed-Mode C-C18 Monolithic Spin Column Extraction and LC–MS/MS

Organophosphates and carbamates are pesticides whose acute toxicities are caused by inhibition of acetylcholinesterase. A liquid chromatography–mass spectrometry/mass spectrometry method was developed and validated for the quantification of 16 organophosphate and carbamate insecticides in human serum. Nonpolar and polar pesticides were simultaneously extracted from serum samples using a simple and fast monolithic spin column procedure using mixed-mode C-C₁₈ cartridges. Recovery was achieved in the range 11.9–99.2 %. Chromatography was carried out on an InertSustain^® C₁₈ HP 3 μm analytical column with gradient elution. Mass spectrometric analysis was performed using an Agilent 6410 Triple Quad Tandem mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The assay was validated over a large concentration range and the limits of detection for all compounds ranged from 0.1 to 50 ng mL⁻¹. The precision for both intra- and inter-day determination of all analytes was found to be acceptable (< 12.9 %) and no significant matrix effect was observed. The developed method was effectively applied to clinical samples from patients presenting at hospital with symptoms of acute intentional organophosphate or carbamate poisoning, demonstrating its applicability to diagnostic applications.

     

Hydrophilic interaction liquid chromatography/positive ion electrospray ionization mass spectrometry method for the quantification of perindopril and its main metabolite in human plasma

A validated method based on liquid chromatography/positive ion electrospray–mass spectrometry (LC-ESI/MS) is described for the quantification of perindopril and its active metabolite, perindoprilat, in human plasma. The assay was based on 500-μL plasma samples, following solid-phase extraction using Oasis HLB cartridges. All analytes and the internal standard (trandolapril) were separated by hydrophilic interaction liquid chromatography using a SeQuant Zic-HILIC analytical column (150.0?×?2.1 mm i.d., particle size 3.5 μm, 200 Å) with isocratic elution. The mobile phase consisted of 10% 5.0 mM ammonium acetate water solution in a binary mixture of acetonitrile/methanol (60:40, v/v) and pumped at a flow rate of 0.10 mL min^?1. Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 5.0–500.0 ng mL^?1 for perindopril and perindoprilat. Intermediate precision were found less than 3.5% over the tested concentration ranges. A run time of less than 6.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method is the first reported application of HILIC in the analysis of angiotensin-converting enzyme inhibitors and can be used to quantify perindopril and perindoprilat in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Determination of cocaine and metabolites in hair by column-switching LC-MS-MS analysis

A method for rapid, selective, and robust determination of cocaine (CO) and metabolites in 5-mg hair samples was developed and fully validated using a column-switching liquid chromatography–tandem mass spectrometry system (LC-MS-MS). Hair samples were decontaminated, segmented, incubated overnight in diluted HCl, and centrifuged, and the diluted (1:10 with distilled water) extracts were analyzed in positive ionization mode monitoring two reactions per analyte. Quantifier transitions were: m/z 304.2→182.2 for CO, m/z 290.1→168.1 for benzoylecgonine (BE), and m/z 318.2→196.2 for cocaethylene (CE). The lower limit of quantification (LLOQ) was set at 0.05 ng/mg for CO and CE, and 0.012 ng/mg for BE. Imprecision and inaccuracy at LLOQ were lower than 20 % for all analytes. Linearity ranged between 0.05 and 50.0 ng/mg for CO and CE and 0.012 and 12.50 ng/mg for BE. Selectivity, matrix effect, process efficiency, recovery, carryover, cross talk, and autosampler stability were also evaluated during validation. Eighteen real hair samples and five samples from a commercial proficiency testing program were comparatively examined with the proposed multidimensional chromatography coupled with tandem mass spectrometry procedure and our reference gas chromatography coupled to mass spectrometry (GC-MS) method. Compared with our reference GC-MS method, column-switching technique and the high sensitivity of the tandem mass spectrometry detection system allowed to significantly reduce sample amount (×10) with increased sensitivity (×2) and sample throughput (×4), to simplify sample preparation, and to avoid that interfering compounds and ions impaired the ionization and detection of the analytes and deteriorate the performance of the ion source.     

Simultaneous determination of six hydrophilic ethers at trace levels using coconut charcoal adsorbent and gas chromatography/mass spectrometry

The main objective of the following study was to determine the efficiency of a method that uses coconut charcoal as a solid-phase extraction (SPE) adsorbent in order to simultaneously detect six hydrophilic ether species in water in the low microgram-per-liter range. The applied method was validated for quantification of ethyl tert-butyl ether, 1,4-dioxane, ethylene glycol dimethyl ether (monoglyme), diethylene glycol dimethyl ether (diglyme), triethylene glycol dimethyl ether (triglyme) and tetraethylene glycol dimethyl ether (tetraglyme). SPE followed by gas chromatography/mass spectrometry of the extracts using the selected ion monitoring mode allowed for establishing low detection limits in the range of 0.007–0.018 μg/L in ultrapure water and 0.004–0.020 μg/L in environmental samples. Examination of the method accuracy and precision resulted in a recovery greater than 86.8 % for each compound with a relative standard deviation of less than 6.6 %. A stability study established a 5-day holding time for the unpreserved water samples and extracts. Finally, 27 samples obtained from surface water bodies in Germany were analyzed for the six hydrophilic ethers. Each analyte was detected in at least eight samples at concentrations reaching 2.0 μg/L. The results of this study emphasize the advantage of the method to simultaneously determine six hydrophilic ether compounds. The outcome of the surface water analyses augments a concern about their frequent and significant presence in surface water bodies in Germany.     

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater

Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry.

Simultaneous Determination of Major Flavonoids in Rat Plasma by a Rapid Resolution LC-ESI–MS Method After Oral Administration of the Flavonoid Fraction of Flos Lonicerae japonicae

A rapid resolution liquid chromatography-tandem mass spectrometric method was developed for the simultaneous analysis of four flavonoids in rat plasma. Key features of this method include a 6.5 min separation by a rapid resolution liquid chromatography system with a 4.6 × 50 mm, 1.8 μm particle size reverse phase column and detection by electrospray ionization tandem mass spectrometric performed in MS–MS mode for multiple reaction monitoring. The developed method was validated with respect to linearity, accuracy, precision and stability and applied for a pharmacokinetic study successfully after oral administration of the flavonoid fraction of Flos Lonicerae japonicae.     

Simultaneous chromatographic analysis of photoinitiators and amine synergists in food contact materials

Photoinitiators (PIs) are components of UV-cured inks widely used in printing of food packaging. These substances can migrate into food and may be a hazard to human health. High-performance liquid chromatography with diode-array detection (HPLC–DAD) has been used for analysis of PIs and amine synergists in food packaging. Analysis was performed with a Kromasil C18 column (250 mm?×?3.2 mm i.d., 5 μm particle size) with a binary mobile phase gradient prepared from acetonitrile and Milli-Q water. The flow rate was 0.5 mL min^?1. The method enables separation of fourteen PIs and amine synergists in a single run. The method was validated for linearity, repeatability, and limits of detection and quantification. Excellent sensitivity (LODs?≤?1.56 μg dm²) and appropriate repeatability (RSD (n?=?10) <0.9 %) were achieved. Different types of food packaging material including plastic films, cardboard, and cans were analyzed and PIs were detected in 47 % of the samples tested (n?=?17). Positive samples were confirmed by use of LC–MS–MS in positive electrospray ionization (ESI) mode.     

Determination of Primaquine in Whole Blood and Finger-Pricked Capillary Blood Dried on Filter Paper Using HPLC and LCMS/MS

Primaquine (PQ) is the only 8-aminoquinoline antimalarial drug in clinical use because of its unique action on hypnozoites and gametocytes of Plasmodium species. We report here simple, sensitive and specific assay methods for the determination of PQ in human whole blood and dried blood spot (DBS) samples using high-performance liquid chromatography and liquid chromatography-mass spectrometry, respectively. Sample preparation was performed by a single or two-step liquid-liquid extraction with organic solvents. For whole blood analysis, separation was obtained on a reversed-phase C18 column with the mobile phase consisting of 0.25 % diethylamine and acetonitrile (7:3, v:v) running at a flow rate of 1.0 ml min^?1. UV detection was at the wavelength 263 nm. For DBS analysis, separation was obtained on a reversed-phase column with the mobile phase consisting of methanol and 0.1 formic acid (1:3, v:v) running at a flow rate of 0.5 ml min^?1. The selected ions generated by electrospray ionization were detected using mass spectrometer. Good precision and accuracy (both within-day and day-to-day assays) were obtained at the concentration ranges under investigation. Limits of quantification for PQ were accepted as 25 ng ml^?1 using 500 μl whole blood and 5 ng ml^?1 using 80 μl DBS samples. The mean recoveries for PQ and internal standard pyrimethamine (PYR) for both whole blood and DBS were over 70 %. The methods were successfully applied for a clinical pharmacology study of PQ in patients with Plasmodium vivax. Excellent correlation (r ²  = 0.997) was observed between the analysis of PQ in paired whole blood and DBS samples.     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 μg/kg by ELISA which correlated to >10 μg/kg by LC-MS/MS.     

Analytical determination of specific 4,4′-methylene diphenyl diisocyanate hemoglobin adducts in human blood

4,4′-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4′-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography–high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.